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5-2012

Sassafras Tea: Using a Traditional Method ofPreparation to Reduce the CarcinogenicCompound SafroleKate CummingsClemson University, kecummi@g.clemson.edu

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Recommended CitationCummings, Kate, "Sassafras Tea: Using a Traditional Method of Preparation to Reduce the Carcinogenic Compound Safrole" (2012).All Theses. 1345.https://tigerprints.clemson.edu/all_theses/1345

SASSAFRAS TEA: USING A TRADITIONAL METHOD OF PREPARATION TO

REDUCE THE CARCINOGENIC COMPOUND SAFROLE

A Thesis

Presented to

the Graduate School of

Clemson University

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Forest Resources

by

Kate Cummings

May 2012

Accepted by:

Patricia Layton, Ph.D., Committee Chair

Karen C. Hall, Ph.D

Feng Chen, Ph. D.

Christina Wells, Ph. D.

ii

ABSTRACT

The purpose of this research is to quantify the carcinogenic compound safrole in

the traditional preparation method of making sassafras tea from the root of Sassafras

albidum. The traditional method investigated was typical of preparation by members of

the Eastern Band of Cherokee Indians and other Appalachian peoples. Sassafras is a tree

common to the eastern coast of the United States, especially in the mountainous regions.

Historically and continuing until today, roots of the tree are used to prepare fragrant teas

and syrups. These traditional uses can be found across cultures throughout its range.

Products made from sassafras are banned from the market by the US Food and Drug

Administration (FDA) due to a carcinogenic compound, safrole, found in the unprocessed

root. Low levels of safrole are permitted in Europe due to the small concentration found

in common spices, including nutmeg and cinnamon. However, in sufficient doses, safrole

causes genotoxicity and cell toxicity, oxidative stress, and liver cancer in laboratory rats

after ingestion. In this study, traditionally prepared tea and the FDA method of

eliminating safrole were analyzed using high-performance liquid chromatography. These

two methods were compared for effectiveness of eliminating or reducing safrole levels.

The FDA method resulted in little to no safrole content. A smaller amount of safrole was

present in the traditionally prepared tea compared to agitate samples, used to gauge a

baseline concentration of safrole present in the root. Collaborations with the Center for

Cherokee Plants within the Cherokee community will help us to return the results of the

research and contribute resources emphasizing the cultural and historical importance of

Sassafras albidum.

iii

DEDICATION

This work is dedicated to my mother, the strongest and most loving person I

know. Also for my father, for his generosity and humor.

iv

ACKNOWLEDGMENTS

I would like to thank my major advisor, Dr. Karen Hall, first and foremost. Thank

you for your unending support and dedication to not only this project, but also for helping

me become a well-rounded individual. This project would not have been possible without

your patience, wisdom, and guidance through every step. Dr. Feng Chen, thank you for

your advice and support, as well as the generous use of your laboratory to complete the

chemical analysis. Thank you Dr. Christina Wells, for helping with the propagation trials

and planting of sassafras in North Carolina. Special thanks to Sarah McClellan-Welsh

and Kevin Welch for everything you have done, including hosting and assisting with the

tree planting project, GIS project, and sassafras tea conversations. Thank you for your

kindness, hospitality, and understanding. To Dr. Chen’s lab, especially Nina Huang and

Greg Jones, thank you for teaching me everything you knew about chemical analysis and

very basic chemistry. Thank you to Alyssa Palmer-Keriazakos, my summer intern, for

being such a cheerful, independent, and patient student of GIS and allowing me to bring

the SPRI experience full circle. I would like to acknowledge the Highlands Biological

Station for the grant which funded my summer field research, and to the Highlands-

Cashiers Land Trust, especially Kyle Pursel. Last but not least, thank you to the people

who took time out of their day to talk to me about sassafras tea.

v

TABLE OF CONTENTS

Page

TITLE PAGE .................................................................................................................... i

ABSTRACT ..................................................................................................................... ii

DEDICATION ................................................................................................................ iii

ACKNOWLEDGMENTS .............................................................................................. iv

LIST OF TABLES ......................................................................................................... vii

LIST OF FIGURES ...................................................................................................... viii

CHAPTER

I. INTRODUCTION ......................................................................................... 1

II. LITERATURE REVIEW .............................................................................. 3

Project Proposal ....................................................................................... 3

Eastern Band of Cherokee Indians ........................................................... 4

Ecology of Sassafras albidum ................................................................. 6

Safrole .................................................................................................... 10

Laboratory Studies ................................................................................. 12

Toxicity and Dosage .............................................................................. 16

Safrole in Food and Spices .................................................................... 19

Previous Studies of Foods Containing Safrole ...................................... 20

Traditional Knowledge & Implications for Chemical Analysis ............ 22

III. TRADITIONAL KNOWLEDGE OF SASSAFRAS TEA.......................... 25

Introduction ............................................................................................ 25

Traditional Harvesting, Processing, and Preparation ............................. 25

Field Methods: Harvesting ..................................................................... 27

Field Methods: Processing ..................................................................... 30

Field Methods: Preparation .................................................................... 32

Results and Discussion .......................................................................... 35

IV. CHEMICAL ANALYSIS USING HPLC ................................................... 36

vi

Table of Contents (Continued)

Page

Introduction ............................................................................................ 36

Materials and Methods ........................................................................... 37

Results .................................................................................................... 47

Observations and Discussion ................................................................. 48

Reduction of Safrole during Boiling ...................................................... 53

V. WORKING WITH NATIVE COMMUNITIES:

EDUCATION AND OUTREACH ......................................................... 55

Introduction ............................................................................................ 55

Working with Native Communities ....................................................... 55

Collaborating with the Cherokee ........................................................... 57

VI. SUMMARY ................................................................................................. 61

APPENDICES ............................................................................................................... 64

A: Propagating Sassafras albidum .................................................................... 65

B: Petitioning the Food and Drug Administration ............................................ 71

C: Reference of Previous Laboratory Studies Using Safrole ........................... 73

D: Abbreviations ............................................................................................... 98

LITERATURE CITED .................................................................................................. 99

vii

LIST OF TABLES

Table Page

3.1 Site and tree characteristics for Sassafras albidum trees harvested in

Highlands, North Carolina.. ................................................................... 31

4.1 Table of samples, extraction methods, and purpose of sample type in

preparation of HPLC analysis.. .............................................................. 41

4.2 Results of HPLC analysis in parts per million (PPM)

for 235nm UV detection. ....................................................................... 44

4.3 Milligrams and ppm of safrole per 300 mL of aqueous solution. ................ 45

4.4 Milligram of safrole hypothetically consumed by a 60 kg person............... 46

C.1 Review of studies using traditional or modern methods of preparation ...... 74

C.2 Review of studies using laboratory animals and bacterial studies

to test the toxicity of safrole and derivatives ............................................... 75

C.3 Review of studies using chemical analysis of foods containing safrole ............... 94

viii

LIST OF FIGURES

Figure Page

2.1 Heterophyllic leaves of Sassafras albidum .................................................... 9

2.2 Chemical Structure of Safrole ...................................................................... 13

3.1 Traditionally prepared sassafras tea ............................................................. 28

3.2 Segments of dried sassafras root chopped into small pieces. ...................... 33

3.3 Sassafras root being actively boiled while preparing

the traditional method samples. ............................................................. 34

4.1 Safrole standard (5,000 ppm), detected at 235 nm with an elution

time of 37.775 minutes.. ........................................................................ 42

4.2 Standard Curve for 235 nm absorption. ....................................................... 43

5.1 Stand of twenty sassafras trees planted at the Center for

Cherokee Plants in Bryson City, North Carolina. .................................. 60

5.1 Transplanted and pruned sassafras sapling .................................................. 66

1

CHAPTER ONE

INTRODUCTION

Sassafras albidum is a North American species primarily known for the tea made

from its roots and root bark. Sassafras has been in use for centuries by the Cherokee,

Chippewa, Creek, Delaware, Iroquois, Seminole, and other Native American tribes

within the East Coast range of the tree (Moerman 2011; Hamel & Chiltoskey 1975).

Many people who were raised in the Southeast remember drinking sassafras tea as a

child, and families in the mountainous regions still make root tea and syrup today. The

Eastern Band of Cherokee Indians in Western North Carolina is one such community that

have preserved and passed down the traditional knowledge needed to utilize sassafras.

The essential oil extracted from the root of sassafras contains 80-90% safrole

(Carlson & Thompson 1997). Safrole is a phenylpropanoid, a type of aromatic compound

that most likely accounts for the strong smell associated with the root (Kamdem & Gage

1995; Rasch 1998). Safrole is found in small quantities in a number of species consumed

on a daily basis, including cinnamon, ginger, nutmeg, and cocoa (Heikes 1994). On the

commercial market, sassafras extract was a common ingredient in foodstuffs, cleaning

products, and cosmetics as a flavoring agent and fragrance. Until 1960, the sale of food

containing safrole was legal up to 20 ppm (Carlson & Thompson 1997). In 1960, the

United States Food and Drug Administration (FDA) banned the presence of the

compound safrole in foodstuff. Experiments had demonstrated the hepatocarcinogenetic

(liver cancerous) effect of large doses of safrole on laboratory rats (CFR, Sec. 189). It is

2

possible to buy safrole-free extracts of sassafras tea concentrate where the safrole has

been removed according to processes set by the FDA (CFR, Sec. 172).

Many studies have demonstrated the carcinogenic effects of high doses of safrole

on laboratory rats, but all these studies have used pure safrole standard (Jin et al., 2011;

Liu et al., 1999; Ueng et al., 2005). As of yet, no research has tested the traditionally

prepared sassafras tea for carcinogenic properties. In fact, no research has investigated

the potential concentration of safrole in traditionally prepared sassafras tea. This research

is important because many people, particularly within southeastern mountain

communities, continue to prepare and consume products made from sassafras.

The purpose of this study is twofold: first, to quantify the amount of safrole

present in traditionally prepared sassafras tea. We will do this by running chemical

analysis of tea samples and samples prepared following FDA guidelines through high

performance –liquid chromatography (HPLC). The purpose of running both the FDA and

traditional samples is to quantify the effectiveness of both methods at reducing safrole

content. The second objective will focus on education and outreach projects, to return the

results of the chemical analysis to the community, with which we collaborated and

complete projects that highlight the cultural and historical importance of sassafras.

3

CHAPTER TWO

LITERATURE REVIEW

Project Proposal

The topic of this research project was suggested during a meeting with

community and tribal members connected to traditional agriculture, history, and granting

agencies of the Eastern Band. They believed researching the toxicity associated with

sassafras products would be relevant and interesting to members of the tribe, as tea and

syrup are commonly made and exchanged between Cherokee members. The products

could eventually contribute to the economy of the Cherokee if the sale of products was

achieved or desired. The banning of sassafras products on the market has raised concern

among those that consume sassafras tea, syrup, and other foodstuffs made from Sassafras

albidum. Although many continue to consume these products, there is a feeling of

hesitancy and uncertainty in conversation about the use of sassafras. For the present,

quantifying the concentration of the main carcinogenic compound in sassafras products

would be meaningful for many who have consumed sassafras their entire lives. With the

information from these results, consumers could make informed decisions on the personal

use of sassafras. Though already a culturally important plant, a study on this species

would support the continued emphasis of sassafras in the culture and honor the traditional

knowledge associated with its use.

4

Eastern Band of Cherokee Indians

The Eastern Band of Cherokee Indians (EBCI) of Western North Carolina own

more than 56,600 acres of land in five counties slightly south and adjacent to the Great

Smoky Mountains National Park (Lambert 2007). There are approximately 13,000

enrolled members, about 8,200 of which live on EBCI-owned land called the Qualla

Boundary (Hall 2006; Lambert 2007). Historically, the Cherokee territory covered about

25.6 million acres in the mountains of the southeast, primarily on the Savannah,

Hiwassee, and Tuckasegee rivers, with their principle town, Echota, on the bank of the

Little Tennessee. The Kituwah settlement, now known as the Kituwah mound, is located

along the Tuckasegee River near Bryson City, North Carolina and was possibly the

original capital of the Cherokee (Duncan and Riggs 2003; Hudson 1979).

Written history about the Cherokee began in 1540 with the exploration of De Soto

and other Spanish explorers. As the French and Spanish settled along the coast and

slightly inland in the 1500’s and 1600’s, there was only occasional contact between the

groups. Some scholars estimate these early settlers brought diseases that killed 95% of

Native Americans within the first century and a half of European contact (Duncan and

Riggs 2003). Being on the edge of the territory claimed by early European settlers, the

Cherokee were caught up in the politics and bloodshed of the Colonial Period, the French

and Indian War, and the Revolutionary War, as well as the slave trade of several ethnic

groups. Exploring settlers slowly absorbed the piedmont regions of Cherokee land and

the tribe became concentrated in the southern Appalachian Mountains (Finger 1984).

5

The “Civilization Policy” of 1789 by the new American government was a

crossroads for the Cherokee in terms of adapting to European-style schools, government,

churches and economy. The written language developed by Sequoyah in 1819, which

may have been a response to these pressures. The Treaty of New Echota of 1835, signed

without permission of the Cherokee people as a whole, gave all Cherokee land east of the

Mississippi to the United States. In 1838, the US government forced 17,000 Cherokee to

territory in Arkansas and Oklahoma on a march now known as the Trail of Tears. About

one-quarter to one-half of the ill-equipped travelers perished on the march (Finger 1984).

A group of three to four hundred Cherokee hid in the mountains of Western North

Carolina to evade capture by US soldiers. They, along with Cherokee that returned to the

territory, were the foundation for the present day Eastern Band of Cherokee Indians

(Duncan and Riggs 2003).

It is important to know this brief history of the Eastern Band when considering the

contemporary Cherokee Indians and the efforts they have made to reclaim their rights and

traditional knowledge. Many historians, ethnobotanists, and anthropologists have studied

and written about the Cherokee, including James Mooney in the 19th

century (Mooney

1992). He was followed by Frans Olbrechts, who worked with the healer Swimmer to

publish a manuscript in 1932, and then by John Witthoft, who published articles from the

1940s to 1970s. There have been a number of books written on the ethnobotany of the

Cherokee in latter half of twentieth century, including authors William Banks (1953),

Myra Jean Perry (1974), David Cozzo (2004), and Karen C. Hall (2006). The Museum of

the Cherokee Indian Press publishes the Journal of Cherokee Studies, as well as many

6

books on the history and culture of the tribe with authors like Barbara Duncan, Vernon

Crowe, and Duane H. King (MCI 2012).

Today, tribal members continue their cultural traditions including the lacrosse-like

stickball game, the Cherokee language, and traditional Cherokee arts and crafts, all of

which are integrated with contemporary festivals. They cater to tourists through

managing a casino, interpretive performances of the Trail of Tears, and selling traditional

arts and crafts. The Cherokee worldview is represented throughout the landscape where

they live, including street signs written in Cherokee language and the preservation of

culturally and spiritually important locations like the Kituwah mound. The Cherokee

language is reinforced through language immersion schools for members. Many families

continue to pass traditional ecological knowledge of plant uses through generations and

between members.

Ecology of Sassafras albidum

Sassafras is a native deciduous tree species of Eastern North America known for

its brilliant fall color and a distinct fragrance. Leaves are bright to deep green in the

summer and change to scarlet, orange, or yellow in the autumn. The tree has

heterophyllic foliage with three leaf shapes: entire, mitten (both right- and left-handed),

and trilobed, as seen in Figure 2.1. On rich sites in the Smoky Mountains, sassafras is

typically a medium-sized tree with a straight bole reaching heights of 9 to 18 m (30 to 60

ft). On the edges of the habitat range sassafras tends to be shrubby. Sassafras is a

dioecious species whose female plants produce small racemes of yellow flowers in March

7

or April (Griggs 1990). The odor of the root is very distinct; the scent has been described

as ‘sweetshop’ or spicy (Dugan 2011).

Sassafras is found along the Atlantic coast (zones 4 to 9) of the United States

from northern Florida to Canada, and west to Michigan, Illinois, and Arkansas (Cullina

2002; Rasch 1998). Sassafras is a generalist species, occupying both full sun and partial

shade sites with a variety of soil types and moistures. The tree can be found in a wide

range of ecosystems, including many species of pine dominated forests and oak-hickory,

maple-beech-birch, and aspen-birch forests. The rapid growth is achieved through a deep

taproot as well as shallow, laterally growing root system that send up root suckers

(Griggs 1990). White-tailed deer, woodchucks, black bears, and rabbits all browse the

leaves during the summer and winter. Many types of birds (wild turkeys, pileated

woodpeckers, and northern bobwhites) and some small mammals eat the ripe fruit, a

single-seeded drupe with a high energy value (Sullivan 1993). Those who wish to

propagate sassafras usually need to stake out the tree for the highly favored drupe (See

Appendix A for Propagation techniques). Sassafras is an important host plant for many

species of moths and butterflies, particularly the host-specific spicebush swallowtail

butterfly (Carter et al., 1999; Sternberg 2004).

Sassafras is typically considered shade intolerant, though it can adapt to low light

intensities of an understory with soil pH of around 5 (Bazzaz et al., 1971). S. albidum is

typically subdominant but can attain dominance by forming thickets on poor soils

through allelopathic activity, which includes the release of terpenes and

phenylpropanoids as well as other secondary defense compounds that deter pathogens

8

and herbivores (Bisset 1994; Gant et al., 1975; Sullivan 1993). These adaptations are

important when considering the chemistry of the allelopathy: the strong presence of

allelopathic compounds are found throughout the tree but mainly concentrated in the

roots. Root wood is porous with characteristic pits and oil cells (Bisset 1994). These

allelopaths include: 2-pinene, 3-phellandrene, eugenol, safrole, citrol, and s-camphor

(Griggs 1990). The sassafras root cortex contains 6-9% essential oil, which consists of

the compounds safrole (80-85% of the total oil), safrole camphor (3.25%),

methoyleugenol (1.1%), tannins (sassafrid), resin, wax, mucilage, sugar, and sitosterol.

Another one percent of the oil includes pinene, eugenol, apoil, 5-methoxyeugenol,

elemincine, estragol, and myristicin (Rasch 1998).

9

Figure 2.1: Heterophyllic leaves of Sassafras albidum

10

Safrole

Safrole (5-(2-propenyl)-1,3-benzodioxole) is a phenylpropene, part of the

aromatic phenylpropanoid family. Safrole has a benzene ring flanked on either side by a

dioxolane ring and a terminal, highly reactive methylene group, as seen in Figure 2.2

(Wink et al., 2008). Safrole is insoluble in water but mixes readily with chloroform,

ether, and other non-polar organic solvents (Budavaris 1989; Burdock 1997). Many of the

aromatic rings that produce smell and taste of plants are derived from the

phenylpropanoid metabolism through the shikimic acid pathway (Dewick 1997; Wink

2010). Phenylpropanoids secondary compounds associated with the taste and smell of a

plant and therefore are used as deterrents against herbivores that shy away from strong

secondary metabolites. The compounds also protect against ultraviolet radiation, fungi,

and bacteria, and can act as pollinator attractants (Hahlbrock 1989). Secondary

compound activity increases in nutrient-poor soils, which is why sassafras, known for its

ability to inhabit roadsides and abandoned fields, would have high allelopathic and

defense tendencies in these locations and therefore higher concentrations of associated

secondary compounds (Ibrahim 2001).

Due to its pleasant smell, sassafras oil used to be a common ingredient (up to 20

ppm) in foodstuffs, cleaning products, and cosmetics as a flavoring agent and fragrance

(Carlson and Thompson 1997). Then on December 3, 1960, the US FDA banned the

presence of safrole, oil of sassafras, isosafrole, and dihydrosafrole in food after

experiments showed a hepatocarcinogenic effect on laboratory rats (CFR, Sec. 189).

Many common spices, including black pepper, cocoa, mace, nutmeg, cinnamon, tarragon,

11

star anise, fennel, parsley, basil, bay laurel, dill, pimento (allspice), and cloves, contain

trace amounts of safrole (Heikes 1994; Zhou et al., 2007). Safrole is also detectable in the

range of 3-5 mg/L (ppm) in soft drinks that use the spices listed above, including the

well-known beverages Coca-Cola and Pepsi (Choong and Lin 2001). Alcoholic

beverages contain between 0.15 and 3 mg/L (ppm) (Curró et al., 1987). In a 2001

document, the European Commission allowed, “1 mg/kg in foodstuffs and beverages, 5

mg/kg for alcoholic beverages with more than 25% alcohol by volume and 15 mg/kg for

foods containing mace or nutmeg” for both safrole and isosafrole (Carlson & Thompson

1997; SCF 2002; SCF 2003). In 2005 the Council of Europe listed safrole as an Active

Principle 1, or suspected weakly carcinogenic substance; no maximum daily limit can be

set for these compounds, but they should be set as low as possible. Safrole is currently

under evaluation by the Council of Europe (CD-P-SP 2005). Sassafras was most likely

targeted because of its high concentration of safrole compared to other spices, including

the often cited nutmeg, which has 2.46 % (w/w) compared to sassafras root (89% w/w)

(Curró et al., 1987).

Pure safrole oil is still imported to Europe, Japan, and the United States from

Brazil and countries in the South Pacific. Safrole-rich species in the Cinnamomum and

Ocotea genera are harvested to be synthesized into piperonyl butoxide, used in pesticides,

and piperonal, a flavoring and perfume ingredient (Miglierini 2008; Oltramari et al.,

2004). Safrole is also an essential ingredient in the production of the illegal drug MDMA,

also known as Ecstasy (Rasch 1998). Safrole is designated as a List I Chemical by the

U.S. Drug Enforcement Administration, implying that the chemical is used in the

12

manufacture of controlled substances as well as having legitimate uses (CFR, Sec.

1310.02).

Laboratory Studies

Safrole is classified as a weak carcinogen in laboratory rodents due to the creation

of safrole intermediates that form hepatic DNA adducts (Liu et al., 1999). Conventional

genotoxicity tests, including sister chromatid exchange and micronucleus tests, tested

positive for in vitro toxicity of safrole, and many in vivo tests of safrole have established

carcinogenic doses of safrole, both through incorporating safrole into the diet and

injection (Jin et al., 2011; SCF 2002). Safrole is absorbed passively from the

gastrointestinal tract, but it is thought that safrole is non-toxic in its unaltered form.

Rather, safrole must be metabolically activated to be toxic to humans, a fact confirmed by

neutral or negative results of an Ames test of safrole (Frohne & Pfander 2004; Swanson

et al., 1979; Wink 2008). Some research has investigated utilizing the toxicity of safrole

for human advantages, including anticancer drugs, insect fumigants, contact pesticides,

and anti-fungicides (Casida et al., 1966; Catalán et al., 2010; Huang et al., 1999; Khayyat

2011; Khayyat & Al-Zahrani 2011; Kim & Park, 2008; Zhao et al., 2005).

Metabolites of safrole formed in vivo include but are not limited to

1’-hydroxysafrole (considered the most toxic), dihydrosafrole (p-n-propyl-

methylenedioxybenzene), isosafrole (1-propenyl-3,4-methylenedioxybenzene), and

eugenol (4-allyl-2-methoxyphenol) (Heikes 1994). As the body attempts to rid itself of

the non-nutritive lipophilic molecule through the liver, the defense system undergoes a

phase I reaction to convert the molecule to a nucleophilic substance (Shibamoto et al.,

13

Figure 2.2: Chemical Structure of Safrole

14

2009). Metabolic activation of safrole to its carcinogenic derivatives can be simplified

into four [4] different transformations. The first [1] transformation involves the oxidation

of the allyl side chain in cytochrome P450 by the enzyme CYP2A6 to form 1’-

hydroxysafrole. This compound can undergo sulfation to form 1’-hydroxysafrole sulfate

(Daimon et al., 1997/8; de Vries 1997; Jeurissen et al., 2004; Zhou et al., 2007). These

electrophilic, sulfuric acid esters form safrole-DNA adducts in human hepatoma (HepG2)

cells and induce cancerous formations (Liu et al., 1999; Miller et al., 1983; Zhou et al.,

2007). Safrole-DNA adducts lead to the induction of sister chromatid exchanges and

chromosomal aberrations, which lead to mistakes in DNA replications and mutations that

has the possibility of carcinogenesis, as well as cytotoxicity (Daimon et al., 1997).

Adducts concentrate in hepatic DNA as the liver is the detoxifier and metabolizer of

drugs for the body (Nakagawa et al., 2009).

A second [2] transformation falls within a different pathway to chemical

carcinogenesis: oxidative stress, which causes faulty incorporation during DNA

replication. Safrole can undergo cleavage of the dioxolane ring to form hydroxychavicol

(4-allyl-1,2-dihydroxybenzene), shown in the pivotal Benedetti study to be the major

urinary metabolite of rodents and humans. The Benedetti et al., study is the only research

as of yet to test the results of human subjects ingesting safrole (Benedetti et al., 1977; Liu

et al., 1999). Hydroxychavicol, best known in connection to betel quid chewing, has the

potential to transform to the reactive elecrophiles ortho-quinone or para-quinone

methide. These metabolites can further transform to reactive oxygen species that can

induce oxidative damage. Hydroxychavicol is more toxic than safrole and has been

15

linked to mitochondrial dysfunction (Bolton et al., 1994; Nakagawa et al., 2009). Though,

unlike DNA-safrole adducts, oxidative stress has been shown to be reversible. The

damage was repaired within 15 days after safrole was administered, compared to stable

DNA adducts that have been detected 30 to 140 days after ingestion (Gupta et al., 1993;

Liu et al., 1999). The damage initiated by hydroxychavicol can also be prevented in vivo

by antioxidants like vitamin E (Liu et al., 1999).

The first two transformations are the main metabolic pathways in animals. A third

[3] transformation involves epoxidation of safrole by the double bond of the propenyl

group to form safrole-2’,3’-epoxide (de Vries 1997). The fourth [4] transformation is the

gamma oxidation of the allylic side chain leading to a carboxylic acid, which can

conjugate with glycine (CSF 2002). The two major safrole-DNA adducts for this

transformation are N2-(trans-isosafrol-3’-yl)2’-deoxyguanosine and N

2-(safrole-1’-yl)2’-

deoxyguanosine (Gupta et al., 1993).

The effect of safrole on laboratory rats and mice is well documented. Rodents

given large doses of pure safrole in their diet (via oral gavage, subcutaneous injection, or

mixed into food) suffered from mortality and a range of liver and kidney damages.

Physiological aliments included slight impairment such as weight loss, mild anemia, and

growth retardation. Aliments also included moderate and severe damage, including liver

tumor masses and nodules, liver enlargement, focal to chronic nephritis in the kidney and

liver, bile-duct proliferation, fatty metamorphosis, and mortality (Abbott et al., 1961;

Daimon 1998; Epstein et al., 1970). Genotoxicity assays were positive for sister

chromatid exchange (SCE), unscheduled DNA synthesis (in cultured rat hepatocytes),

16

chromosomal aberrations, gene mutation, and cell transformation (Howes et al., 1990;

Ishidate & Sufuni 1985; Mihr et al., 1985; Purchase et al., 1978). Safrole did test neutral

or negative in an Ames test (Salmonella reverse mutation assay) as mentioned before, as

well as mouse dominant lethal assay, bone-marrow micronucleus assay, and unscheduled

DNA synthesis in HeLa cells (Baker & Bonin 1985; Epstein et al., 1972; Gocke et al.,

1981; Martin et al., 1978; Mirsalis et al., 1982). Ingestion of safrole at high doses can

cause symptoms such as nephritis, unconsciousness, weakness, liver disturbance, CNS

stimulation, and skin irritation (Wink 2008). Also see Appendix C for complete chart of

laboratory studies.

Toxicology and Dosage

Toxicology research utilizes two models to determine the potency of compounds,

including qualitative and quantitative methods. Qualitative methods can include

observations of lesions and clinical signs, while the quantitative model examines the dose

and exposure levels. The ultimate goal is to determine a structure-activity relationship

that outlines the biological effects of a substance at physiological and biochemical levels.

To evaluate a compound for human safety, toxicology studies examine several species of

laboratory animals (rats, mice, dogs, rabbits, primates) that have similar physiology and

genetic structure to humans (Reagan-Shaw 2007). Researchers use several dose levels,

where the higher dose levels are to understand the biochemical mechanisms and a

maximum amount is established for safe consumption. The mode of ingestion or

exposure should be considered in clinical tests; for example, feeding laboratory rats the

17

food additive compounds through their diet as opposed to subcutaneous injection or oral

absorption (Dixon 1976).

As the 16th

century Swiss-German chemist/physician Paracelsus held, ‘Poison is

in everything, and no thing is without poison. The dosage makes it either a poison or a

remedy’ (“Paracelsus” 2012). In estimating toxicity and the possibility of carcinogenicity,

the dose is critical as is the perceived risk. For risk in using food additives in particular,

no amount of carcinogen is accepted as additions to food are risks than can be avoided

(Extoxnet 2012). In terms of dose, dose translation is necessary to compare doses

between different species, particularly between human and laboratory animals. The use of

body weight to compare doses between studies has been criticized as it may not

accurately correlate to volumes of blood and plasma proteins (Dixon 1976). Body surface

area (BSA) has been shown to associate these parameters, or total blood and plasma

protein volume, in addition to basal metabolism, renal functions, and oxygen and calorie

utilization. The equation used by Reagan-Shaw et al. (2007) to translate doses between

animal species and human:

Human equivalent dose (mg/kg) = Animal dose (mg/kg) * Animal Km/Human Km

(Reagan-Shaw et al., 2007). Km values (mg/m2) are listed in the paper for several species

and are based on height-weight measurements. In this thesis, mg/kg bw (body weight)

refers to mg of compound by body weight of the subject, either human or animal. The

unit listed as mg/kg refers to mg of a compound in kg of material.

Another aspect of dosage to consider is the difference in employing high versus

low dosage. The ‘Virtual Safe Dose’ for human consumption is calculated with a linear

18

model, “which assumes that cancer causation is directly proportional to dose and that

there are no unique effects of high doses” (Ames & Gold 2000). A dosage margin of

safety for humans is typically calculated by dividing a lethal dose of the compound for

laboratory rodents by a factor of 100 (Segelman et al., 1976). An exaggerated high dose

of a compound, as in experiments to understand the mechanisms of carcinogenicity, can

cause wounding and death of tissue and cells and chronic cell division of neighboring

cells. It is possible the resulting cellular necrosis and carcinogenesis is inflated in high

doses, a point discussed in literature on safrole.

The Benedetti et al. (1977) study examined the response of rats and mice to

consumption of safrole in addition to human subjects. Even at the smallest dose of 0.63

mg/kg bw (body weight), 88% of the safrole was eliminated by the rats within 24 hours.

The authors point out that when the dose of safrole for the rodents was increased to levels

common in carcinogen studies (between 500 to 1000 mg/kg bw), safrole rapidly

accumulated in the liver and kidney, and safrole was not metabolized as quickly and

therefore eliminated more slowly than rats fed smaller doses. They argue that the toxic

accumulation associated with these carcinogenic studies was due to this slow metabolism

of safrole and subsequent tissue absorption was a result of the unrealistic doses. Lower

doses (or accurate amounts of safrole in food and drinks) would not lead to safrole

accumulation in tissue muscle (Benedetti et al., 1977). Gupta (1993) found a dose of400

mg safrole/kg bw for mice to be the point of saturation of detoxification enzymes.

19

Safrole in Food and Spices

The HERP Index, or Human Exposure/Rodent Potency index lists a daily level of

safrole ingestion at 1.4 mg/kg bw per day for a 60 kg person from the various safrole-

containing spices like black pepper, star anise, cumin, cinnamon, and ginger root (HERP

2011). The European Commission estimated the daily intake of consumers (by the

quantity of safrole added to food by industry) to be 0.3 mg/day, or 0.005 mg/kg bw for a

60 kg person (SCF 2002). The WHO estimates a daily range of safrole intake as 4 to 569

μg for Americans and between 0.6 and 879 μg for those in the European Union, based on

spice content and annual volume consumed by country (WHO 2009). The median toxic

dose (TD50) of safrole, according to the HERP Index, for mice is 51 mg/kg bw per day

(HERP 2011). Segelman et al. estimate a dose of 0.66 mg/kg bw may be toxic to man,

based on a study by Epstein et al. (1970) that found 66 mg/kg bw of safrole administered

over a 21 day period to be carcinogenic to infant male mice. Studies commonly use a

margin-of-safety factor of 100 to estimate toxicity, hence the 0.66 mg/kg bw (Segelman

et al., 1976).

In the only study found with human subjects, Benedetti et al. (1977) observed the

effects of human consumption of safrole with two doses (0.163 mg and 1.655 mg). They

concluded both amounts of safrole were completely eliminated in 24 hours with little

possibility of safrole accumulating in tissue muscles. In the human subjects, safrole was

eliminated as small amounts of unchanged safrole and safrole metabolites, specifically

1,2-dihydroxy-4-allylbenzene, eugenol, and an isomer of eugenol. The metabolites 3’-

hydroxylisosafrole and 1’-hydroxysafrole (the most toxic metabolite) were not detected.

20

The authors suggest the absence of the latter metabolites could also be due to the dose or

to the differences in metabolism between man and rodent (Benedetti et al., 1977).

Safrole is present in the flowers of Piper betle, used in the betel quid chewing

tradition (similar to tobacco dip in the United States) prevalent among the male

population of Taiwan. Betel quid has been linked to the high rates of oral cancer due to

the high level (15,000 mg/kg plant material) of safrole present in the flowers, which are

used in the betel quid mixture. Exposure to safrole in the saliva during chewing is

estimated between 70 mg/L to 68120 mg/L (or 420 μmol/L) (Chen et al., 1999; Liu et al.,

2000). Research on betel quid chewing in India and Southeast Asia suggested the

presence of safrole could be causing the high rates of oral cancer and other diseases by

inhibiting the bactericidal activity and releasing reactive oxygen species (Hung et al.,

2003). A study examining betel quid chewing among pregnant women found that adverse

pregnancy outcomes, including lower birth weight, stillbirth, fetal malformation, and

premature delivery, were 2.8 times higher than women who did not chew betel quid

(Yang et al., 2001). Overall, it is difficult to compare betel quid chewing to consuming

sassafras tea due to the different paths of ingesting safrole (absorbed through the mouth

and saliva versus gastrointestinal absorption) and the range of doses.

Previous Studies on Foods Containing Safrole

A handful of studies have examined sassafras tea. Carlson & Thompson (1997)

examined several types of herbal products derived from sassafras, including leaves, tea

concentrate, herbal powder capsules, and tinctures, as well as a ‘sassafras tea’ prepared

21

from instructions from a modern herbal guide. The ‘tea’ made in this study was infused,

or the tea bag steeped in hot water for thirty minutes as opposed to the traditional method

of decocting, or boiling, the whole or roughly chopped root. They demonstrated that

herbal products of sassafras ranged from zero to a miniscule amount of safrole in tea

made from the leaves, to containing 92.4% safrole in sassafras oils. The ‘tea’ they

prepared had safrole content ranging from 0.03 to 1.37 mg per gram of powdered root

material, or only 6.9% to 17.2% of total safrole present in the root (Carlson & Thompson

1997). There were several differences between the Carlson & Thompson study and this

research, including the use of root bark powder instead of traditionally harvested and

processed root and root bark. The researchers also infused the root bark powder in hot

water instead of the decoction method that is used by all the consultants in this study.

Another study by Heikes (1994) quantified the amounts of known carcinogenic

compounds in commercial, un-brewed tea mixes, without actually heating the material.

Both of these studies (Carlson & Thompson and Heikes) focused on finding a more

efficient, cost-effective method of analysis than the official method adopted by the FDA

in the 1960’s. Kapadia et al. (1978) did not prepare tea per se, but did extract in an

aqueous solution using sassafras root bark. The researchers used petroleum ether,

methylene chloride, and ethanol to extract the oil (which would include the safrole), and

then injected 15 mg of the safrole-free ethanol extract in rats (Kapadia et al., 1978).

22

Traditional Knowledge & Implications for Chemical Analysis

Traditional knowledge, especially in Native American communities, comes from

a cumulative, inter-generational custom of passing along practices, beliefs, ideas, and

wisdom. It is transmitted through cultural behaviors and generated by close contact with

the natural environment. It encompasses every part of a society: politics, history,

anthropology, and philosophy (Huntington 2005). The inherent complexity in natural and

human social systems is recognized and categorized with the assumption that humans are

a part of the web. Balance in natural systems requires reciprocity and respect. Knowledge

is indigenous or traditional when “the meanings as well as the categories of sense making

are generated internally within a cultural community”, as well as a physical environment

(Gupta 2010; Viergever 1999).

Within the system of traditional knowledge, traditional medicine is less a remedy

or cure, and more of a way of balanced well-being. The World Health Organization

defines it as, “the sum total of knowledge, skills and practices based on the theories,

beliefs and experiences indigenous to different cultures that are used to maintain health,

as well as to prevent, diagnose, improve or treat physical and mental illnesses” (WHO

2008). When traditional medicine is taken out of context of its originating culture, it can

be termed alternative or complementary medicine. Without the history or tradition to

sustain them, medicines can be prepared or used differently from their original intention,

sometimes leading to harmful results. This is not to say that traditional knowledge

regarding plant preparation is invalid, only that if the product is of poor quality, taken

inappropriately, prepared differently, or used at the same time as other medicines, the

23

product can be ineffective or malignant (WHO 2008). The traditional knowledge and

particular species are often targeted, as in the case of sassafras tea, rather than if and how

the knowledge was taken out of cultural context.

People that use sassafras today look at the extended use of sassafras through

history as proof of its safety. Steve Brill, a wild food forager in New York, states “People

have enjoyed sassafras in moderation for thousands of years with no ill effects and

consumed it in root beer before it was replaced by artificial chemicals. I think sassafras is

safe” (Brill 1994). A home remedy book, or do-it-yourself herbal, says, “Everyone knows

that sassafras has been used for centuries as a spring tonic. Try it, it’s delicious”

(Williams 1998). Unfortunately, there are instances of injury from traditional methods

prepared incorrectly. For example, a case study published in the Journal of Postgraduate

Medicine describes an elderly woman who complained of diaphoresis. The doctor traced

the excess sweating and hot flashes to her recent habitat of consuming ten cups of

sassafras tea a day, as instructed by a family member (Haines 1991). The article does not

indicate how strongly she prepared the tea or exactly how many ounces she consumed

from ten cups. Another case study from 1987 reports a 47-year-old woman ingesting a

teaspoon, or 5mL, of sassafras oil and immediately vomiting, followed by trembling

(Grande & Dannewitz 1987).

There have been many studies highlighting the importance of traditional methods

of preparation, including several on safrole-containing species. This research closely

followed the study of Reynertson et al. on a bark tea made from Cinnamomum

carolinense, a study that specifically examined the differences in extracting the bark

24

using alcohol versus water (2005). Research in Egypt studied safrole-containing spices

collected in local markets for the degradation of safrole during common cooking methods

(Farag & Abo-Zeid 1997). A wild ginger species (Asarum spp.) used in traditional

Chinese medicine was analyzed before and after a one-hour decoction (Chen et al., 2009).

The kava-kava controversy is well known in the health food circle for multiple cases of

severe liver damage when the roots are extracted with ethanol or acetone rather than the

traditional method of maceration in water and coconut milk (Singh & Devkota 2003;

Whitton 2003).

It is important to remember as well that plant medicine is most likely derived

from secondary compounds produced for protection against herbivores, pathogens, fungi,

and other modes of attack. Plants have evolved a myriad of defense mechanism, from

neurotoxin effects that can cause confusion or sedation to physiological effecting,

including growth suppression or organ damage. These secondary compounds, often very

toxic to predators, can be harnessed by humans for beneficial effects (Briskin 2000).

The purpose of this research was not to discredit or devalue the traditional

knowledge that informed this study. Native American cultures have been using sassafras

for centuries, along with other plant species with deadly and toxic compounds. As of the

conclusion of this study, there have been no confirmed, direct linkages between drinking

sassafras tea and harm reported in any CDC Morbidity and Mortality Report or the FDA

MedWatch program (CDC 2012; MedWatch 2012). These programs are the monitoring

methods used by the FDA to follow any adverse, unexpected, or unusual results of

consuming FDA-regulated products (Love 1999).

25

CHAPTER THREE

TRADITIONAL KNOWLEDGE OF PREPARING SASSAFRAS TEA

Introduction

The methods and materials used in this study of harvesting, processing, and

preparing sassafras root are based on discussions with enrolled members of the EBCI and

non-Cherokee persons. These people have made sassafras tea for much of their lives and

many were taught as children by their parents or grandparents. They were consulted on

their methods of making tea in order to create a specific formula for tea-making to be

used as the ‘standard’ in the laboratory, a process that is replicable and uses specific

quantities. The ‘standard’ method represents a typical way to prepare tea and was a

compromise between slight differences among the processes. The harvesting, processing,

and preparing techniques varied with the consultants, including the age and habitat type

of the sassafras tree, how the root was processed for tea and storage, the proportion of

root to water in making the tea, and the length of time the root was boiled and steeped.

Traditional Harvesting, Processing, and Preparation

The processes included on harvesting, processing, and preparing sassafras root

represent one way of demonstrating how decisions were made for this research and do

not imply the totality of processes or people who use sassafras. Many consultants

harvested saplings because they are abundant, easier to dig up, and are considered weedy

by some homeowners. Some consultants dug from both young and mature trees. A few

26

indicated they dug roots in certain seasons, including only in late fall, winter, or early

spring, as these were when the plant energy was concentrated in the root. The processing

step was the most variable among root harvesters: some consultants dried the root spread

on newspaper, while others hung them in bags. The consultants either chopped the root

into chunks of various sizes and weights, whittled the root lengthwise into strips, or kept

the smaller, thinner roots whole. In making the tea, the root was boiled from 10 minutes

up to an hour, removed from the heat source, then let steep for a few hours, overnight, or

up to 24 hours.

There were several points of general agreement among the consultants. They all

emphasized the sustainable harvest of the root as to not to harm the larger trees, or only

harvesting saplings if they are considered weedy or in a site about to be bulldozed. Every

consultant indicated that they left the root bark on the roots, washed the root very

thoroughly after harvesting, and hung the root in breathable containers, most commonly a

mesh onion bag. Breathable containers are essential to keep the dried root from molding.

The consultants agreed that dried root would last many years, though typically it was

used in one-year period due to steady use.

All the consultants agreed on the proper color of the tea, though many found it

difficult to describe: light brown, almost see-through, yellowish-brown, medium honey-

colored, or the color of amber beer (See Figure 3.1). Any lighter and it would be too

weak tasting, and any darker it would overpower the senses. Many described the amount

of root to be used in a pot or single cup of tea in terms of handfuls, as in a double handful

of root per kettle of water or one chunk of root for a mug of water. The art of making

27

sassafras tea is in the experience and practice of knowing how strong the root smells as to

how strong the tea will be, and checking on the steeped tea for the color and taste. The

amount of root and water and how long it is boils and steeps is secondary to the color,

smell, and taste. The more root you use, the faster it will steep, while the less root used,

the longer you steep it.

Tea is made with either fresh root or dried root, and there seemed to be no

preference for either type. Dried root needs to be rehydrated in order to release the flavor,

which can be done by boiling the root once, letting it steep overnight, and boiling again

the next morning and let steep again, or by letting boil and steep for a longer period of

time. For the amount of tea to drink, the consultants were all in agreement. If a person

was sick, they should have about three cups of tea, otherwise one to two cups a day is

enough. Tea is typically consumed in the fall, winter, and spring, especially in the spring

months when people are liable to get sick or prepare for the changing season. Fresh tea is

best, but if a big pot is made it can be kept in the refrigerator for up to a week.

Field Methods: Harvesting

All roots were dug within two weeks during July 2011 on property managed by

either the Highlands Biological Station or the Highlands-Cashiers Land Trust. All the

land was within Highlands, North Carolina, a small town in the southern plateaus of the

Blue Ridge Mountains. Many of the harvesting sites were around 3,800 feet in elevation,

with one site at 4,100 feet. Sassafras trees grow prolifically at this elevation and habitat,

which is classified as temperate rainforest (Chamber of Commerce 2011).

28

Figure 3.1: Traditionally prepared sassafras tea.

29

A total of nine trees were harvested for an average of 40 grams of root material

per tree. The terrain shape index, landform index, and diameter at breast height (dbh)

were measured for each of the trees, and the habitat type was described. The roots were

removed with hand shears, and the soil samples were collected from around the

excavated roots, to a depth of six inches. Soil samples were analyzed by the Clemson

University Agricultural Services Lab for general soil properties and nutrients. With the

exception of one site (Site 7), all the primary nutrients (nitrogen, potassium, and

phosphorus) were in the low medium to low range recommended for adequate plant

growth. The soils in all the sites were moderately to extremely acidic, with an average

soil pH of 4.23 ± 0.6 (optimum soil pH for plants is 5.8 to 6.5). See Table 3.1 for

complete descriptions of harvest trees and sites.

Positive root identification was achieved by following a large surface root three to

four feet from the tree bole. The surface root sometimes dipped into the soil, where it was

followed by tunneling along the root. Identification was also attained by olfactory

identification of the excavated root, which smells characteristically of ‘sweetshop’, candy

store, or ‘spicy’ when fresh. The two young saplings that are included in the sampling

were dug up completely and the entire root system up to the main root of the colony was

collected. Only trees approved by the Highlands-Cashiers Land Trust or the Highlands

Biological Station were harvested.

The size and shape of the roots is most likely dependent on the age of the tree, site

conditions like soil type and slope, and size of the colony, if part of one. Observations

were made that the older trees had much thicker, darker roots, almost a deep reddish-

30

brown color, with potent smelling roots that smelled characteristically like a sweet-shop

and earthy. Soil surrounding older roots had a very strong sassafras smell. Young

saplings, three or four years old, had roots that were much lighter in color with a sharper,

sweeter smell then the older trees without the associated smell in the soil. Surface roots,

or roots typically acting as suckers in a colony, were very pliable and thinner compared to

non-surface roots.

Field Methods: Processing

The roots were washed thoroughly (carefully keeping the root bark) in tap water

and weighed on a Mettler H51 balance scale with 0.01 g precision. The length and

diameter were measured with a digital caliper. The roots were dried on paper for a

minimum of 48 hours to prevent mold growth, and then placed in separate brown paper

bags until prepared for samples. The bags kept in an open box in a well-ventilated room

until they were boiled for tea.

When the roots were dry, they were weighed and measured again. The roots had

an average moisture loss of 61% when air-dried, or ranging between 51% to 67%

moisture loss. One 4 g sample was dried in an oven for 6 hours, or until the dried weight

did not fluctuate, where it lost a further 9% moisture, for a total of approximately 70%

moisture difference between dried and fresh root.

The air-dried dried roots were cut into segments that each weighed from 0.1 g to 1

g approximately (See Figure 3.2). The segments were mixed together in equal parts, 10 g

from each tree, so each tree root represented one-ninth of the total weight. When root

31

Site # Height

(feet)

DBH

(inches)

Habitat Slope Soil

pH

1 17 3.6 Acidic upland with Rhododendron

and Vaccinium, part of a colony,

sandy loam soil, understory

North facing, slight

slope

3.4

2 12 1.8 Acidic upland with Rhododendron

and Vaccinium, part of a colony,

sandy loam soil, understory

Southwest facing,

slight slope

3.9

3 15.5 2.2 Acidic upland with Rhododendron

and Vaccinium, part of a colony,

sandy loam soil, understory

Southwest facing,

slight slope

4.5

4 <6 <1 Acidic upland with Kalmia,

Rhododendron, and Vaccinium,

Rocky, sandy soil, understory in a

windy, exposed mountainside

Southwest facing,

very steep

4.1

5 17 2.2 Oak-hickory forest, understory

and part of a colony

Southwest facing,

slight slope

4.3

6 57.5 28.8 Acidic soil with dense covering of

Rhododendron and Kalmia,

roadside

South facing, steep

slope.

4.1

7 <6 <1 Sapling on disturbed site, roadside South facing, slight

slope

4.9

8 49 12.5 Old-growth, acidic upland forest

with Rhododendron. Is dominant

in the canopy with no colony

West facing, some

slope

3.6

9 <6 <1 Sapling in open, disturbed site

with full sun. Part of a colony of

saplings

South facing, no slope 5.3

Table 3.1: Site and tree characteristics for Sassafras albidum trees harvested in

Highlands, North Carolina.

32

segments were selected for preparing the tea, the bag was shaken thoroughly and root

segments were chosen blindly and at random. Oven-dried root segments were not used in

tea preparation as someone would not normally dried the root in the oven before making

tea.

Field Methods: Preparing

The traditional method of preparing tea was standardized to consistently produce

a cup of sassafras tea with approximately the same color and smell each time. This was a

subjective observation, but acceptable for this study as tea is consumed with a wide range

of color and smell intensities. Just as a variety of tree age classes and habitats are

harvested, a variety of preferences exist within tea preparation. As the consultants drank

tea within this spectrum of tea intensities, we intended to cover the range of tea consumed

to reflect any differences in safrole content. Once a standard time of boiling, steeping,

and re-boiling was established, the standard method did not change to accommodate

changes in tea color.

The roots were boiled twice: once to rehydrate the roots, which were then left to

steep ‘overnight’, then boiled again to make the tea. The water and root are heated

together to boiling (when the water started visibly bubbling), which marked the

beginning of the boil time. The heat source is turned down to ‘medium’ to simmer for 15

minutes (See Figure 3.3). The tea was removed from the heat source and left to steep

‘overnight’ for 12 hours without refrigeration. The second boil was performed as the first,

or the root and water brought to a roiling boil together, with the time beginning at first

33

Figure 3.2: Segments of dried sassafras root chopped into small pieces.

34

Figure 3.3: Sassafras root being actively boiled while preparing the traditional method

samples.

35

signs of boiling. The tea was allowed to actively boil for 20 minutes and removed from

the heat source. The root segments were used once for each replicate of each sample.

Results and Discussion

The standardized method described in this section was based on conversations

with people knowledgeable in the use of the species. This standardized method,

particularly in the preparation steps, is as similar to the traditional method as could be

replicated in a laboratory setting (using distilled water, hot plates, and laboratory

glassware instead of kitchenware). Future studies might look closely at the traditional

knowledge of sassafras, including the range of harvesting, processing, and preparation

methods.

36

CHAPTER FOUR

CHEMICAL ANALYSIS USING HPLC

Introduction

The objective of this chemical analysis was to quantify the amount of safrole

found in both the method outlined in the FDA regulation and the traditional method of

preparing sassafras tea. A third sample set, the agitate samples, was added to measure the

concentration of safrole present in the root without degrading the safrole with heat

extraction. The samples were run through a high-performance liquid chromatography

(HPLC) system using a reverse-phase C18 column method. The FDA used gas

chromatography in the 1960’s to detect and quantify safrole, but the developments in

sensitivity and reliability of HPLC lead us to use this method (CFR Sec. 189; Larry

1970). Also, many recent papers have used HPLC to quantify safrole, including a paper

by Reynertson et al., 2005 from which we adapted the methodology for the chemical

analysis. This paper was used as a model because the study was recent, so it utilized

modern analytical techniques, and because the research closely paralleled the

ethnobotanical objectives of this project.

Plant material is inherently variable, particularly in the concentration of secondary

compounds. The time of day or season of the year, soil and microclimate, herbivory and

pathogen activity, and age and history of a particular tree can determine specific chemical

quantities. The experiment was designed to encompass the spectrum of harvest and

37

preparation styles, as well as variation among young or mature trees and different habitats

and elevations.

Materials and Methods

Laboratory materials

- Safrole standard (98%) – Chem Service Inc. in West Chester, Pennsylvania.

- ACS-grade MeOH and HPLC-grade ACN – Fisher Science in Georgia.

- Water – deionized

- YMC ODS-AQ S-8 4.6 x 250mm, 5μm, 120Å HPLC column

Traditional Method

Samples 1a, 2a, and 3a (see Figure 4.1 for chart of the samples) were prepared by

boiling 2 grams of air-dried root segments in a 500 mL glass Erlenmeyer flask with 300

mL distilled water, using two hot plate/stirrers (VWR Scientific Products 370 and Fisher

Science). A small piece of aluminum foil was loosely placed over the top of the flask to

reduce water evaporation. The samples were prepared as described in Chapter 3, or

boiled for 15 minutes, let steep for 12 hours, and re-boiled for 20 minutes.

Fifty micrograms (50 μg) of safrole standard was added to the samples 2a, 3a, 2b,

and 3b, following the methods in Reynertson et al. 2005. These spiked samples would

show whether safrole is degraded without complication of root material.

The traditional method samples were run twice due to the inconsistency of the

results in the first run. Each sample set of the traditional methods has six samples

whereas the FDA method and agitate sample set contains three samples each.

38

FDA Method

The FDA samples were prepared based on the generalized methods defined by

regulations. Sassafras extract is the aqueous solution “obtained by extracting the bark

with dilute alcohol, first concentrating the alcoholic solution by vacuum distillation, then

diluting the concentration with water and discarding the oily fraction” (CFR, Sec. 172).

Samples 1b, 2b, and 3b were prepared using ACS grade methanol diluted to 80%

methanol in 500 mL glass Erlenmeyer flasks. Each of the FDA samples were stirred

using a stir plate and stir rod for 35 minutes, or the same boil time of the traditional

method samples. Following the methodology outlined by the FDA, vacuum distillation

was done using a Büchi Rotovap. The heating bath was set at 65°C, the evaporation point

of methanol. The solution was diluted back to 300 mL using distilled water. The solution

was poured into the funnel separator, allowed to settle for 10 minutes, and then run

through the separator until 5 mL remained. The remaining liquid in the funnel separator

represented any sassafras oil found in the root and was not included in the analyzed

sample. The remaining aqueous solution (after removal of the oil fraction) had no

detectable oil, characteristic sassafras scent, or color.

Agitate Samples

The agitate samples were prepared with 10 g of root and 300mL of cool distilled

water. The samples were stirred with a stir plate and stir rod for 50 minutes, let sit for 20

hours, and filtered. The agitate sample went through a longer period of agitation and

steeping until the color of the resulting samples was close to color of the traditional tea

39

samples. The color was not standardized but based on the descriptions provided by the

consultants. A larger quantity of root was added for the same reason. No heat was applied

during any part of the process and the stirring was kept at a low, steady speed as to not

cause excess heat. The resulting concentration was divided by five to compensate for the

increased amount of root but no correction was taken for the increased time of agitation.

HPLC Analysis

All samples were filtered through 0.2 μm syringe filters prior to analysis. Analysis

was performed on a Shimadzu Prominence UFLC (Ultra-Fast Liquid Chromatography)

system using a reverse-phase C18 column (YMC ODS-AQ S-8 4.6 x 250mm, 5μm,

120Å). Results were monitored at 235 and 254 nm using UV detection. The mobile phase

consisted of two solutions, (solvent A) distilled water and (solvent B) acetonitrile (ACN).

The HPLC conditions were as follows: linear gradient of 5% (B) at 5 minutes, 15% (B) at

10 minutes, and 100% (B) at 45 minutes for 10 minutes, with a stop time of 55 minutes.

The injection volume was 10 μL. Retention time for safrole standard was 37.77 ± 0.23

minutes (See Figure 4.1).

The Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated

based on the blank determination method cited in Sanagi et al., 2009. LOD is the lowest

concentration of a compound that can be detected, but without the need to specifically

quantify the concentration. LOQ is the lowest quantifiable concentration identified by the

analytical procedure. LOD and LOQ are important analytical validations that define the

limits of the analytical laboratory machine (Sanagi et al., 2009). There are many accepted

40

methods for quantifying LOD and LOQ, including the blank determination method

outlined in Sanagi et al. (2009). The blank determination method was chosen for this

study as the method is applicable when a blank sample returns results with non-standard

deviations. LOD and LOQ is calculated by running at least three blank samples with no

standards or compounds added. The mean concentration and mean standard deviation of

the blank are found. LOD is the concentration of the blank sample plus three standard

deviations (xm + 3Sd) while the LOQ is the analyte concentration of the blank sample plus

ten standard deviations (xm + 10Sd), where (xm) is the mean concentration and (Sd) is the

standard deviation (Sanagi et al., 2009).

LOD and LOQ measured 1.514 ppm and 2.049 ppm for 235nm, respectively.

Triplicate samples of diluted safrole standard of 10, 50, 100, 200, and 1000 ppm were run

to configure a standard curve. Standard curves were calculated for 235 nm absorption,

yielding the following equation which with to calculate the amounts of safrole present in

the samples. For 235nm absorption: the equation was [Area = 628.2326*Concentration],

as seen in Figure 4.2. For the 235nm absorption, the p-values for the intercept variable (or

‘b’ in the equation y = mx + b) were greater than the level of significance, meaning it is

not included in calculating the sample concentrations. The R-squared value for 235nm

absorption was 0.9579. P<0.05 was considered statistically significant. Based on the

absorption spectrum of safrole, the 235 nm wavelength was determined to be a more

stable absorption time and therefore was the only wavelength reported in these results.

41

Number Sample Extraction

Method

Purpose

1a Root Traditional Quantify the presence

or absence of safrole

2a Safrole Standard Traditional No root, to ensure traditional

method eliminates safrole

3a Safrole spiked Root Traditional Quantify amount of degradation

1b Root FDA Quantify safrole to compare to #1a

2b Safrole Standard FDA No root, to compare to #2a

3b Safrole spiked Root FDA Quantify amount of degradation

Agitate Root Cold water To extract unaltered safrole using

water

Table 4.1: Table of samples, extraction methods, and purpose of sample type in

preparation of HPLC analysis. Traditional method samples are signified with the letter ‘a’

after the sample number while FDA method samples have the letter ‘b’ after the sample

number.

42

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 min

0

50

100

150

mV

Detector A Ch1:235nm

1.1

94

4.2

65

6.4

52

14.7

85

16.8

46

18.1

8018.5

10

19.0

08

19.8

26

20.5

4221.0

70

21.3

8321.5

67

22.1

35

22.4

14

22.7

5923.4

25

23.7

9324.0

12

24.3

33

24.6

25

24.8

31

25.3

08

25.5

13

26.5

1526.8

24

27.3

2527.5

70

27.8

0428.2

60

28.5

6028.8

99

29.1

06

29.4

68

30.1

7530.3

72

30.6

9230.8

25

31.0

1131.1

58

31.3

1731.4

58

31.6

7331.8

50

32.1

8832.3

42

32.4

9232.8

03

32.9

8233.1

42

33.2

7533.5

95

33.8

2034.1

74

34.5

0134.9

78

35.8

52

36.4

76

36.8

17

37.7

75

39.0

5739.4

09

39.6

7139.8

19

40.0

50

40.6

01

40.9

1941.1

96

41.7

0841.8

79

42.2

4042.7

53

43.2

67

43.4

74

43.7

8944.1

29

44.6

5645.1

26

45.2

9145.5

73

45.9

9946.1

79

46.4

2446.6

53

46.9

7847.2

08

47.3

9247.6

59

48.3

9548.6

63

48.8

66

49.1

33

49.4

6249.6

27

49.8

5350.0

20

50.2

61

54.5

39

54.8

92

61.9

15

62.7

53

Figure 4.1: Safrole standard (5,000 ppm), detected at 235 nm with an elution time of

37.775 minutes.

43

Figure 4.2: Standard Curve for 235 nm absorption. The equation generated by the linear

regression: Area = 610.03*Concentration. Triplicate runs of 10, 50, 100, 200, and 1000

ppm of safrole standard are indicated by red open circles. Upper and lower 95%

confidence intervals represented by dotted lines.

44

Sample PPM Mean

(PPM) Std Dev

1a-1 7.206

1a-2 0

1a-3 9.055

1a-4 8.917

1a-5 6.198

1a-6* 35.36 6.275¹ 3.707

2a-1 0

2a-2 1.854

2a-3 0

2a-4 0

2a-5 0

2a-6 0 0.309 0.757

3a-1 26.09

3a-2 13.80

3a-3 18.36

3a-4 5.880

3a-5 9.653

3a-6 0 12.30 9.258

1b-1 0

1b-2 0

1b-3 0 01 0

2b-1 0

2b-2 0

2b-3 0 0 0

3b-1 0

3b-2 1.93

3b-3 0 0.644 1.12

Agitate-1 21.25

Agitate-2 24.12

Agitate-3* n/a* 22.691 2.033

Table 4.2: Results of HPLC analysis in parts per million (PPM) of safrole at 235nm. The

mean and standard deviation of each of the sample sets is listed.

*Samples 1a-6 and Agitate-3, were removed from the sample sets as the results were

outliers or due to a contaminated column. The concentration was determined by the

calibration curve. 1Samples 1a are significantly greater than the samples 1b and significantly less than

Agitate 1 & 2 samples based on student t-tests at α=0.05

45

Sample Average

Safrole in mg/ 300 mL

Std. Dev.

Average Safrole in ppm or mg/L

Std. Dev.

1a 188.2 111.2 627.5 370.7

2a 9.27 22.7 30.9 75.7

3a 368.9 277.7 1229.7 925.8

1b 0 0 0 0

2b 0 0 0 0

3b 19.3 33.5 64.48 11.67

Agitate 680.6 61.0 2268.7 203.3

Table 4.3: Milligrams and ppm of safrole per 300 mL of aqueous solution.

Concentrations calculated from Table 4.2.

46

Sample Safrole in mg/ kg Std. Dev.

1a 3.14 1.85

2a 0.15 0.38

3a 6.15 4.63

1b 0 0

2b 0 0

3b 0.32 0.56

Agitate 11.34 1.02

Table 4.4: Milligram of safrole hypothetically consumed by a 60 kg person.

Concentrations calculated from Table 4.3.

47

pH of Traditionally Prepared Tea

The pH of three separately prepared, traditional tea samples was quantified using

a pH probe. The probe was rinsed with distilled water, inserted into the tea sample, and

rinsed again with distilled water. The traditionally prepared tea samples were prepared

following the methods listed above.

Results

Concentrations in ppm of the HPLC analysis of the traditional, FDA and agitate

samples can be seen in Table 4.2. One sample prepared using the traditional method with

root only (1a-6) was discarded as an outlier with a large Cook’s distance. Sample labeled

Agitate-3 in Table 4.2 was discarded due to contamination of the column.

The methods outlined in the paper by Reynertson et al., 2005 were followed in

preparing comparative samples, namely the spiked samples (2a, 3a, 2b, & 3b). The

samples 2a and 2b were prepared with 50 μg safrole standard while the samples 3a and

3b were prepared with root and 50 μg safrole standard, as outlined in Table 4.1. When

calculating the concentration of safrole standard added to the samples, the amount

suggested by Reynertson et al. (2005), it was discovered that the amount was too small to

be detected by the HPLC system. In a 300 mL solution, 50 μg contributes a concentration

of 0.67 mg/L, or 0.67 ppm. The LOD and LOQ for this system were 1.514 ppm and

2.049 ppm. Unfortunately, these samples could not be used in discussion of the results of

the chemical analysis.

The agitate samples were intended to provide a baseline amount of safrole found

in the root and extracted with water. We compared the agitate samples to the traditionally

48

prepared teas to observe how the quantity of safrole differs between samples being

actively heated and boiled and those samples simply being agitated. Although the agitate

samples were agitated longer than the traditional methods, the samples were prepared

based on color just as a person making sassafras tea will judge the readiness of the tea on

its color. The elevated amount of root in the agitate sample was compensated by dividing

the concentration by five, as five times as much root was used. It was interesting to note

that while the agitate samples did not have the characteristic sassafras smell, but they did

have a woody, earthy smell that was not unpleasant.

Based on a student t-test, the concentrations of the traditional method samples

were significantly greater than the FDA method samples (P <0.05) and significantly less

than the agitate samples (P<0.05) based on α=0.05.

Observations and Discussion

We found three important points of discussion from the chemical analysis. First,

the FDA method was extremely efficient at removing the safrole oil by agitation using

organic solvents. There was one sample with a small amount (1.878 ppm) of safrole

present, but this is probably due to imperfect laboratory procedures and not a reflection of

the methodology. We observed that the resulting liquid from the FDA method was

odorless and colorless. There was no characteristic sassafras smell associated with the

liquid and therefore, it may be unappealing for a marketable product.

The second observation related to the results of concentrations of the agitate and

traditional method samples, as listed in Table 4.3. The average safrole concentration of

49

the agitate samples was 2268.7 ppm ± 203.3, or 681 ± 61.0 mg of safrole per 300 mL of

aqueous solution. The traditional method of preparing sassafras tea (Sample 1a) resulted

in an average concentration of 627.5 ± 370.7 ppm or 188 ± 111.2 mg of safrole per 300

mL of aqueous solution. Based on these results, the traditional method samples have 3.6

times fewer grams of safrole per cup (300 mL) of tea as compared to agitate.

Since essential oil composes 6-10% of the weight of the root cortex of sassafras,

and safrole composes 80-90% of the essential oil, by taking the highest estimates the root

contains around 9% safrole (Carlson & Thompson 1997; Kamdem & Gage 1995). A cup

of tea, or around 300 mL, is typically prepared with 2 grams of root. We would then

expect the cup of tea to contain 180 mg of safrole oil. The traditional method samples

contained 188 ± 111.2 mg of safrole per cup, or close to the expected concentration. On

the other hand, roots extracted with water by agitation yielded 681 ± 61 mg of safrole per

300 mL of tea. It is difficult to compare this experiment to previous research as the

methods to agitate the sample are going to differ with each study, but it was interesting to

note the difference between the amount of safrole we expected to find and the amount of

safrole extracted by aqueous agitation. The study by Carlson & Thompson (1997)

showed an 88.9% reduction of safrole from the unbrewed teas compared to the brewed

teas. The study found ranges of 0.18 – 16.0 mg/g product in unbrewed teas that were

reduced to 0.09 – 4.12 mg/g product in the brewed teas.

Chemically, it seems unlikely that using a polar solvent (water) to extract a non-

polar solute (safrole oil) should extract more oil than a non-polar solvent (methanol or

another organic solvent). Safrole is miscible in alcohols, not water (Kamden & Gage

50

1995; Sethi et al., 1976). Though, it is difficult to compare the studies as many journal

articles do not detail extraction processes. However, it is possible to say that boiling the

root as in the traditional method aided in reducing the concentration of safrole in the

resulting aqueous solution, as compared to the agitate samples.

The third and last observation relates to the literature on safrole content and

regulations. Returning to the previous calculations, if we expect one cup of tea with 2

grams of root and root bark to have about 180 mg of safrole, a person weighing 60

kilograms would consume 3.0 mg of safrole per cup. This calculation is supported by a

similar estimate by Segelman et al. (1976) who calculated 3.0 mg/kg safrole is found in

one tea bag containing 2.5 g of sassafras bark. We found that sassafras tea has 188 ±

111.2 mg of safrole per cup (300 mL) of tea, as listed in Table 4.3. Therefore, a 60 kg

person would consume 3.13 ± 1.85 mg/kg bw of safrole per 300 mL of tea (Table 4.4). A

person consuming agitated, non-boiled tea would be consuming 11.34 ± 1.02 mg/kg bw

of safrole per 300 mL. The United States FDA does not allow the presence or addition of

safrole to any foods or beverages and the European Commission of the European Union

allows the presence of safrole in foods containing nutmeg and mace only up to 15 ppm.

Traditional tea, according to this study, has around 628 ± 370.7 ppm of safrole per 300

mL (Table 4.3).

Using the Body Surface Area (BSA) dose translation formula discussed earlier, a

60 kg person would consume 3.14 mg of safrole, which is equivalent to a dog consuming

5.8 mg/kg bw. A study by Hagan et al. (1965) conducted a six-year study that

administered safrole to dogs in doses of 5, 20, 40, and 80 mg/kg bw. After six years, with

51

the dogs receiving a tablet six days a week, the dogs experienced microscopic, minimal

focal necrosis, bile-duct proliferation, fatty metamorphosis, and hepatic cell atrophy

(Hagan 1965). Another study with dogs that spanned seven years found liver injury early

in the experiment, but hypothesized that the lack of damage found later in the study was

due to adaptation to the continuous intake of safrole (Weinberg & Sternberg 1966).

The dose equivalent for rats would be 19.4 mg/kg bw. As stated before, many

toxicity studies administer doses in the range of 100 to 1000 mg/kg bw, though Daimon

et al. (1998) tested single doses of 1, 10, 100, 250, and 500 mg/kg bw. Two DNA-safrole

adducts per 107 nucleotides were found after a dose of 10 mg/kg bw, while a 1 mg/kg bw

dose did not produce adducts and a dose of 100 mg/kg bw produced two major and two

minor adducts per 107 nucleotides. Friedman et al. (1971) found that a 10 mg/kg dose of

safrole had no effect on microsomal enzyme activity, which aid in the detoxification

process. Chang et al. (2002) found the metabolic saturation of safrole occurred between

150 and 300 mg/kg for rats, or much greater than the amount of safrole found in a cup of

tea. Saturation of safrole means the body cannot eliminate the compound quickly and the

safrole accumulates in tissue muscle, especially in the liver and kidneys, to increase the

risk of DNA adducts and carcinogenesis (Benedetti et al.,1977).

Based on these few studies, there was no indication that rats or dogs fed safrole at

the amounts listed above developed liver tumors or cancer. There are not enough studies

that have tested these particular doses on laboratory animals to conclude with certainty

that the amount of safrole found in sassafras tea will lead to carcinogenesis. These few

studies are promising, but more research needs to be conducted.

52

Plant secondary compounds, used by the plants for defense against predators and

pathogens, are utilized by humans for the same purpose, to rid the human body of disease

by way of pharmaceuticals (Bourgaud et al., 2001). Secondary compounds must be used

properly, as the same compounds that humans harness to starve off disease can harm the

body. Safrole is a proven hepatocarcinogen to laboratory animals, causing macro- and

microscopic liver lesions, liver tumors, inability to gain weight, and reduced feeding (Jin

et al. 2011; Long et al., 1963; Miller et al. 1983; Taylor et al., 1964). These symptoms

exhibited by laboratory animals make sense when we consider the traditional medicinal

qualities of sassafras tea, including ‘overfatness’ and to thin the blood during the change

of seasons, especially during the spring (Moerman 2011). Considering the laboratory

tests use high doses of safrole compared to the amount of safrole found in traditional tea,

we can hypothesize that the extreme symptoms observed in laboratory animals occur to

some degree in those drinking sassafras tea, but possibly to the human advantage. While

laboratory rats had difficulty gaining weight or had a reduced appetite while consuming

safrole, humans perhaps used small amount of safrole in sassafras tea to help shed winter

pounds. A seven year study of administering safrole to dogs found evidence of liver

injury early in the research but not late in the study. The researchers suggested the dogs

adapted to the continuous intake of safrole and regenerated the damaged liver tissue

(Weinberg & Sternberg 1966).

53

Reduction of Safrole during Boiling

A handful of studies have attempted to explain the process that occurs when

safrole-containing substances are subjected to heat. One study examined the spices star

anise, cumin, black pepper, and common cooking ginger, which contain safrole in the

essential oil, and found that washing, drying, and cooking the spices all reduced safrole to

safe levels. Boiling the spices reduced the safrole content from 955 mg safrole per kg

material in untreated seeds to 375 mg/kg in whole seeds boiled for one minute all the way

to 9 mg/kg in powdered seeds boiled for five minutes (Farag & Abo-Zeid 1997). The

study on a Chinese wild ginger reduced the 1.57 to 2.76 mg safrole per g material to 0.2

mg/g or undetected in most samples after a one-hour decoction of the root (Chen et al.,

2009). Carlson & Thompson (1997) hypothesized that the lower safrole content in their

sassafras ‘tea’ (the tea was infused not decocted) could have been due to either the

decreased solubility of sassafras oil in water or volatilization of the compounds.

Three chemical reactions could be occurring to reduce the safrole content in a cup

of boiled tea. First, the essential oil, including the safrole oil, could be still in the root.

Carlson & Thompson (1997) hypothesized that safrole content was reduced due to the

insolubility of the essential oil in the root, which agrees with their finding of high

recovery of safrole from alcohol- based sassafras oil tinctures. The second situation could

involve volatilization of safrole as it is being heated. The compound has a low vapor

pressure (0.0706 mm Hg at 25°C or 1 mmHg at 63.8°C), making safrole primarily a

vapor at room temperature and pressure (Sigma-Aldrich MDMS S9652). If the compound

was boiled at high temperatures for an extended period of time safrole would have some

54

volatility. Carlson & Thompson (1997) cited volatilization as well as reduced solubility

of safrole in water for the reason safrole content was reduced an average of 88.9% from

unbrewed to brewed tea. Many laboratory studies mention the volatilization of safrole

from safrole-spiked rodent diet, which was left in open in room temperature. One study

includes Homburger et al. (1965) that measured the evaporation rate of safrole to be

11.3% per three day period (Crampton et al., 1997; Huang et al., 1999; Long et al., 1963).

The last possibility is that safrole is being altered or degraded during the boiling

process, as described in the Reynertson et al. (2005) paper. Reynertson et al.

hypothesized that even though safrole is insoluble in water, insolubility is not the issue as

samples spiked with safrole still resulted in no detected safrole. Since there were a

number of degraded by-products after samples were boiled, it is probable that the safrole

was not volatilized. Rather, the safrole was probably degraded during the boiling process

by hydroxylation of the dioxolane ring. The hydroxylation could be due to the slightly

acidic nature of the tea, which they measured at pH 4.4 (Reynertson et al., 2005). For this

research, the average pH for the traditional method tea sample was measured at pH 5.1 ±

0.1, which is considered acidic.

55

CHAPTER FIVE

WORKING WITH NATIVE COMMUNITIES: EDUCATION AND OUTREACH

Introduction

During the research, we have collaborated with the Cherokee community, who

continue to drink sassafras tea according to their traditional method. In addition, there are

many non-Native people that grew up harvesting, processing, preparing sassafras, and

drinking sassafras tea. The Cherokee best represents a cohesive community of people that

maintain the knowledge and tradition of preparing sassafras tea. Since the Center for

Cherokee Plants has worked to maintain and continue traditional knowledge of native,

culturally signification plants, this work might not have been possible.

Working with Native Communities

This research, although not strictly participatory, is based on a model of working

with Native communities through participatory research used by many researchers. In this

model, research, education, and action are combined and research is based on the

circumstances of the community. The researchers work to connect communities with

resources, data, and scientific knowledge of benefit to them. There is an underlying

assumption that communities will benefit from the research as resources are based on an

“ecological give-and-take approach”, where knowledge, time, or mentoring by a

community is returned to them through conscience acts of reciprocity (Davis & Reid

1999; Cotton 1996).

56

Linda Tuhiwai Smith said, “The term ‘research’ is inextricably linked to

European imperialism and colonialism. The word itself, ‘research’ is probably one of the

dirtiest words in the indigenous world’s vocabulary” (1999). Unfortunately, many Native

communities around the world have been exploited by researchers. Some research has

even been harmful, especially research related to data collection and reporting on

negative issues like alcoholism or drug addiction. Such research can lead to communities

being ostracized or stigmatized, with participants feeling betrayed. In the worst cases,

participatory research has left participants feeling invaded, patronized, or inferior, which

is why many Native communities today have created strict rules to how and with whom

research can be conducted on their land (Davis & Reid 1999).

Anthropological and botanical studies in particular are in danger of being of little

use to Native communities, and unfortunately some research on traditional medicines has

been unconcerned with collaboration, effects of publication, or reciprocity. Given the

history of medical, anthropological and archeological research, contemporary research

projects with Native communities absolutely should be a collaborative effort between the

community and the researchers. Christopher (2005) outlines several recommendations for

non-Indian researchers, including involving the community from the formation of the

question to the conclusion, as well as ensuring that the community has access to the

research and results. The research should be based on values, concerns, and interests of

the community and contain culturally-appropriate methodologies (Weaver 1997). The

project should also have clear and immediate benefits for the community. Knowledge for

57

the sake of knowledge, according to activist and historian Vine Deloria Jr., should not be

tolerated by Native Americans (1969).

Collaborating with the Cherokee

Although this project was concerned with the perceived toxicity of products made

from Sassafras albidum, this research did not draw the official support of the tribe due to

the historical use of sassafras for spiritual and medicinal purposes. However, we were

able to work with the Eastern Band of Cherokee Indians Cooperative Extension to

provide a means for education and outreach about the species. The Cooperative

Extension acted as a ‘cultural consultant’ to advise us on the appropriate channels to

distribute the research results (Weaver 1997). We planted sassafras trees and created a

GIS map and pamphlets for the Center for Cherokee Plants (CCP), a project under the

Extension office. The Center for Cherokee Plants, located between Cherokee and Bryson

City, North Carolina and near the Kituwah mound, is a nursery that cultivates and

experiments with Cherokee heirloom varieties of edible crops. They collect, save, and

distribute seed within the tribe to encourage gardening with old varieties and healthy

eating. The Center emphasizes youth and elder participation by establishing community

and school gardens around the Qualla Boundary and projects including the Backyard

Ramp Patch, Native Plant Study, Cherokee Community Greens Patch, and the Cherokee

Farmers Market. They are the hub of agriculture and gardening in Cherokee as they work

with community civic clubs and are connected with youth and elders alike. The Center is

58

a medium to encourage interactions between elders, adults, and youth and the exchange

of traditional and indigenous knowledge (CCP 2011).

By working with the CCP, we were able to hold discussions about sassafras tea

and most importantly, give back to the community. Education and outreach comprise a

significant part of this project for several reasons, including to disseminate scientific

knowledge about the properties and safety of sassafras products and to highlight the

cultural and historical importance of sassafras products. We promoted the use of sassafras

in several ways: first, by planting a stand of twenty trees of sassafras on the property of

the CCP, as seen in Figure 5.1. The CCP can decide in what ways they will use trees,

either by harvesting material for demonstration purposes, allowing tribal members to

harvest material for their own use, or as a physical reminder of how sassafras is a

culturally significant species for the Cherokee. The trees also provide a basis for future

research, ranging from topics on forestry to food chemistry. We also are in the process of

creating pamphlets to be displayed with other educational material at the Cooperative

Extension office in the city of Cherokee as well as at the Center for Cherokee Plants.

Appendix A, focused on simple means of propagating sassafras, will be modified for

these pamphlets. A copy of the thesis will be given to the Museum of the Cherokee

Indian, which is also required for research under the auspices of the Tribal Research

Committee, though we did not submit this research for their approval per their request

(EBCI C.O., Sec. 70-3).

We hosted a high school intern during Summer 2011 to complete a Geographic

Information System (GIS) project to map the nursery grounds of the Center for Cherokee

59

Plants. The GIS map was made into a 3’ x 4’ laminated wall display and also into printale

sheets for record-keeping. The Center asked not for the maps to be either replicated in

print form, other than their own copies, or available on the internet, which we have

agreed not to do. This is an example of how researchers need to be flexible to the wishes

of the Native community which with they work.

The continued use of sassafras trees for traditional foods and beverages could

bring attention to land that is under development on the Qualla Boundary. Many current

stands of sassafras in use by tribal members are open fields or sites slated for bulldozing.

In the future, a non-timber forest product or products could be developed to sell if tribal

members so desired. There could be potential for members of the tribe to petition the

Food and Drug Administration based on this research, but more research and chemical

analysis would most likely be needed before individuals could market and sell sassafras

products. Appendix B contains more information on how to submit a petition to the FDA.

60

Figure 5.1: Stand of twenty sassafras trees planted at the Center for Cherokee Plants in

Bryson City, North Carolina. The three-year-old trees were planted in Spring 2011.

61

CHAPTER SIX

SUMMARY

Sassafras tea is consumed today by many communities in the Southeastern United

States, particularly in the Appalachian Mountains. Products made from the roots of

Sassafras albidum have been banned from the market by the FDA due to the presence of

the confirmed carcinogenic compound, safrole, based on tests using pure safrole standard.

Toxicological studies typically test compounds by administering large doses of the

compound to laboratory animals in order to understand the mechanisms of

carcinogenesis. These studies are needed to understand the mechanisms but should be

carefully analyzed when attempting to discuss the effects of ingesting safrole at daily

levels. Before this project, no research had quantified the concentration of safrole in

traditionally prepared sassafras tea. Additionally, no studied have used traditionally

prepared teas in laboratory rodent cancer potency tests. Even though the number of

people that consume traditional sassafras products is unknown, we can assume that many

people grew up drinking the tea continue to consume it, as well as people that recently

learned through herbal medicine guides. Due to the regular use of sassafras tea, it is

important to understand any potential dangers associated with consuming products

derived from the tree.

This study was informed by traditional knowledge of harvesting, processing, and

preparing sassafras tea, as understood from conversations with members of the Eastern

Band of Cherokee Indians (EBCI) as well as non-Cherokee well-versed in making

62

sassafras tea. The first objective of this study was to conduct chemical analysis using

HPLC. The intention was to compare the FDA method of extracting sassafras oil with a

standardized process of preparing sassafras tea to quantify the amount of safrole present.

An agitate sample was analyzed to compare the influence of heat on the quantity of

safrole. The second objective was to return the results of this study to communities that

utilize sassafras tea, namely the EBCI. We also emphasize the importance of reciprocity

for the Cherokee community, who has dedicated their own resources to this project.

The results of the chemical analysis demonstrated that the FDA method was very

effective at eliminating safrole entirely, as only one sample showed a miniscule amount

of safrole. Additionally, analysis revealed that traditionally-prepared sassafras tea

contains 188 ± 111.2 mg of safrole per 300 mL, or 0.63 ± 0.37 mg safrole per mL of

solution. This is a safrole concentration of 628 ppm in traditionally prepared sassafras

tea. Previous research examining the safrole content in root and root bark predicted a

similar quantity of safrole, or 180 mg per 300 mL. On the other hand, the agitate samples

from this research contained a higher concentration of safrole, or 680 ± 0.061 mg per 300

mL, or 2.3 ± 0.2 mg/mL of solution. This is a concentration of 2269 ± 203.3 ppm, or

more than predicted from previous estimates of safrole concentration in essential oil. As

the agitate samples in this research used water as the extraction solvent (versus organic

solvents used previously), it is difficult to conclude if safrole is reduced in the boiling

process. If we assume the agitate samples in this study are the reference point of safrole

content, then boiling does reduce the amount of safrole found in traditionally prepared

tea.

63

These three methods of extracting sassafras root resulted in important

observations. First, the traditionally made sassafras tea had a pleasant smell and color -

properties that many cultures associate with the tea. Second, the agitate samples did not

elicit the characteristic sassafras smell and it took more root and time to achieve the

desired color. Lastly, the FDA method resulted in a clear, odorless product with

undetectable sassafras odor. In hindsight, the concentration of safrole found in sassafras

tea may be serving some purpose, including simply contributing taste and scent

properties to the tea or functioning medicinally.

In conclusion, this research contributes to a growing body of knowledge, both for

those in the academic world as well as practitioners and caretakers of traditional

knowledge. This research will serve at least two purposes: first, the results of this

research will help people make informed decisions about their personal consumption of

sassafras products. Second and more broadly, this will highlight the importance of

traditional preparation methods when analyzing foods and beverages, especially those

historically used by Native communities.

64

APPENDICES

65

Appendix A

Propagating Sassafras albidum

Sassafras is notoriously challenging to propagate or even find in nurseries. Not

only is it problematic to transplant (due to its deep taproot) but ripe seeds are difficult to

find in the wild (Dirr 1998). The species is dioecious so only female plants produce seed,

which are often eaten quickly by birds and wildlife. Greenish-yellow flowers produce

single-seed drupes on red pedicles that turn a dark blue-purple when mature (Bonner

2012). The species can be propagated from fresh, ripe seeds, dried seeds, cuttings, or root

scions (Rasch 1998).

Transplanting

Sassafras can be grown in a container from seed and the tree transplanted any

time of the year. A tree taken from the wild or from field plantings should be true

seedlings (not root sprouts) that are transplanted in the late winter or early spring. The

trees started by seed and grown in large pots until planting seem to transplant better than

those transplanted directly from a field (Sternberg 2004). The colonal root system of

sassafras, along with its deep taproot, makes it difficult to transplant saplings or trees

easily. Root pruning in the late fall or early winter can encourage a tree to develop a

healthy root ball before it is transplanted in the spring.

When transplanting saplings, the tree should be balled and burlapped in moist,

loamy, acidic, well-drained soil (Dirr 1998). About a third of the plant should be pruned

to encourage root growth (See Figure A.1). If the main bole or branches break, as

66

Figure A.1: Transplanted and pruned sassafras sapling.

67

brittle wood tends to do, then the stem is cut to the ground and a new stem will sprout. A

sassafras thicket is obtained by cutting the stem back to encourage root suckers, but if a

single stemmed tree is desired, the suckers should be removed (Sternberg 2004).

Seeds

Sassafras trees produce seed around 10 years of age (minimum of 4 years old) and

reach maximum production from ages 25 to 50 with seed crops every year or every other

year. Seed viability is around 35% and the seed can remain viable for six years on the

forest floor. A study on Sassafras randaiense (Hayata) Rehder cited inhibitors in the seed

coat and cotyledon that cull germination, requiring removal or scarification of the seed

coat and followed by two to three months of cold stratification (Chen & Wang 1985).

Sassafras seeds do not store well as they are an oily seed (versus a more starchy seed),

with 47% lipid content (Bonner 2012). To break a natural dormancy of the embryo, the

seeds need a cold-wet stratification with a period of 120 days at 41°F (5°C) in moist

sandy or mineral soil (Bonner 2012; Griggs 1990; Haywood 1994). Germination can then

be tested in sand or a germination soil mix at 70° to 85° F. (21-29° C) for up to 120 days.

One method of germinating seed by the Woodlanders Nursery in Aiken, SC is to

sow seed in a ‘community pot’, or a three-gallon container with well-draining potting

soil. The seeds should be rehydrated in water for 24 hours, surface sterilized, and

scattered in a sterilized three gallon pot of soil with a half inch of soil covering the seed.

The community pot should be watered, covered with hardware cloth, and placed outside

for the entire winter until the seeds germinate in the spring. The pot is dumped out and

68

seedlings separated to be potted individually (Bob McCartney, personal communication

November 21, 2011).

Root Cuttings

A study by Sicuranza (2007) showed poor results for sassafras propagation from

stem sprouts, but worthwhile results from root cutting propagation. It has been found that

root cuttings yield plants with longer life spans, less root suckering, and better

architecture than grafted plants (Orndorff 1977). Root cuttings are collected from October

to December, when roots contain the highest percentage of carbohydrates, and placed in a

2:1:1 peat, loam, sand mixture can be planted out successfully (Del Tredici 1995, Dirr

1998). Some suggest taking root cuttings in February when the ground thaws and storing

the roots in dry sand for three weeks before planted in sandy soil. If roots are harvested in

the summer, Sicuranza (2007) showed July as the most successful month.

Evans & Blazich suggest harvesting root pieces that are 5 to 10 cm in length in

early winter. Del Tredici (1995) recommends root pieces that are 10 to 15 cm long for

roots being planted outdoors in late fall or early winter. A straight cut is made on the end

closest to the parent plant while a slanted cut is made on the opposite end (Evans &

Blazich 2011). The roots need to be washed and fibrous roots removed. The root pieces

are placed vertically in a moist medium, preferably pure sand or 1:1 peat to perlite mix

with 10-20% sand. Griggs (1990) recommends placing stem sprouts vertically or larger

roots horizontally. The straight cut, or where the root was closest to the parent plant,

should be covered by 2 to 3 inches of soil (Evans & Blazich 2011). The medium and

69

roots are stored in a cool, dark location for three weeks until put in a warm greenhouse.

There is a danger of water stress as the sudden warmth will cause shoots to develop

before roots (Sicuranza 2007).

Pests & Disease

Foliage disease is the most prominent problem among S. albidum, along with a

strong susceptibility to fire damage. Overall, sassafras is usually free from pests and

disease, probably due to the strong presence of secondary defense compounds, but it does

suffer from the occasional cankers, leaf spots, mildew, wilt, root rot, Japanese beetle, and

sassafras weevil (Dirr 1998).

Propagation Trials

Sassafras seeds were collected in August in Clemson, South Carolina and left to

air dry on newspaper. For these trials, the fleshy seed coats were allowed to dry and then

removed manually, but Bonner (2012) recommends removing the pulpy flesh before

storage or propagation by rubbing the seed over hardware cloth. Two methods of

processing and cold-wet stratifying the seeds were tested. The first method involved

removing the dried seed coat entirely, which can be done by carefully cracking open with

a small knife or fingernail and extracting the round, light brown seed. The seeds were

surface sterilized in a 10% bleach solution for 30 seconds, rinsed, and placed in a

sterilized plastic snap-lid container with damp perlite. The second method leached the

seeds of any chemicals that would inhibit germination. The seeds were placed in

70

cheesecloth like fabric and placed in a back toilet bowl for fourteen days. The seeds were

scraped with sandpaper, surface sterilized, and placed in damp perlite.

All containers were placed in the vegetable drawer of a refrigerator (at 41° F or 5° C) for

four months. In March 2012 the seeds were sown in potting soil and placed outside to

germinate. The seeds will be monitored for success rate of germination of either the

leaching method or removal of seed coat.

A third method of stratifying and germinating the seeds was a method

recommended by Woodlanders Nursery in Aiken, SC, termed ‘community pot’ (personal

communication, Nov. 21, 2011). This method (see above description) was begun for the

experiment but unfortunately had to be terminated. Future studies in sassafras

propagation should strongly consider this method.

The propagations trials are still ongoing. In Fall 2011, a dozen sassafras saplings

considered weedy by the landowner in Cherokee, NC were cut around with a sharp

shovel to encourage a root ball. Six of the trees were transplanted in March 2012 and six

more will be transplanted in Fall 2012 to the Center for Cherokee Plants. The saplings

will be monitored to measure success rate of spring and fall transplanting.

71

Appendix B

Petitioning the Food and Drug Administration

According to the FDA website, the government agency receives about 200

petitions a year (webpage last updated in 2008). The evaluation of a petition can take a

few weeks or up to a year. The majority of the petitions come from industry and

consumer groups, though individuals are allowed to submit petitions. There is a specific

format for citizen petitions that must be followed for submission, which can be found by

searching for Title 21 of the Code of Federal Regulations, Sections 10.30. It contains six

parts: action requested, statement of grounds, environmental impact, economic impact (if

applicable), certification, and identifying information.

The code that specifically targets the use of sassafras bark to make sassafras tea is

[Code of Federal Regulations: Title 21, Volume 3, Sec. 189.180 Safrole].

The full title: Title 21 – Food and Drugs, Chapter I -- Food and Drug Administration,

Department of Health and Human Services, Subchapter B – Food for Human

Consumption (Con’t), Part 189 – Substances Prohibited from Use in Human Food,

Subpart C – Substances Generally Prohibited from Direct Addition or Use as Human

Food. Sec. 189. 180 Safrole.

The code states in part (b), “Food containing any added safrole, oil of sassafras,

isosafrole, or dihydrosafrole, as such, or food containing any safrole, oil of sassafras,

isosafrole, or dihydrosafrole, e.g., sassafras bark, which is intended solely or primarily as

a vehicle for imparting such substances to another food, e.g., sassafras tea, is deemed to

72

be adulterated in violation of the act based upon an order published in the Federal

Register of December 3, 1960 (25 FR 12412)” (CFR, Sec. 189).

Specifically, the petition should outline the confirmed carcinogenic properties of

pure safrole standard and related metabolites in comparison to pivotal papers like

Benedetti et al. (1977) that found a lack of 1’-hydroxysafrole (a carcinogenic derivative

of safrole) in human subjects after ingestion of safrole. Next, the recent research on the

non-toxicity of traditionally prepared foods, beverages, and medicines should be

mentioned (Chen et al., 2009; Farag & Abo-Zeid 1997; Reynertson et al., 2005; Singh &

Devkota 2003; Whitton 2003). The results of this research should be cited as well as any

other future research on the toxicity or carcinogenicity of traditionally prepared sassafras

products.

Summaries of all the studies can be found in the following reviews:

Cropwatch.org/: Safrole

SCF. 2002. Scientific Committee on Food, European Commission: Opinion of the Scientific Committee on Food on the safety of the presence of safrole (1-allyl-3,4-

methylene dioxy benzene) in flavourings and other food ingredients with flavouring

properties. (SCF/CS/FLAV/FLAVOUR/6 ADD3 Final).

WHO 2009. World Health Organization: WHO Food Additives Series: 60. Safety

evaluation of certain food additives. The 69th

Meeting of Joint FAO/WHO Expert

Committee on Food Additives.

73

Appendix C

Reference of Previous Laboratory Studies Using Safrole

Studies on betel quid chewing were included in this chart as they gave the unique

opportunity to study the effects of safrole injection in humans. The inflorescence of Piper

betle contains 15.35 mg safrole when fresh, which metabolize to dihydroxychavicol and

eugenol (both mentioned in the chart) (Chang et al., 2002). Betel quid chewing is vastly

different from consuming sassafras products, both in the complexity and composition of

material and in the mode of safrole absorption. Both products contain safrole, so we can

observe similarities in physiological effects from a range of concentrations.

Many studies quantify the presence of carcinogen- DNA adducts as a risk for

cancer is influenced by the capability of a compound to form these covalent bonds. There

is a correlation between the incidence of carcinogen-DNA adducts and the formation of

carcinogenic tumors, but adducts do not predict the risk of cancer (Groopman & Skipper

1991).

74

Table C.1: Review of studies using traditional or modern methods of preparation. R

ed

uced

Safr

ole

Level

Not

det

ecte

d a

fter

boil

ing

0.3

75 t

o 0

.009 m

g

(in 1

g m

ater

ial)

Mai

nly

undet

ecte

d

to 0

.2 m

g (

in 1

g

mat

eria

l). A

lso

found i

n t

he

trad

itio

nal

Chin

ese

pre

par

atio

n n

o m

ore

than

0.0

2 m

g

0.0

3 t

o 1

.37 m

g (

in

1 g

mat

eria

l)

0.6

09 m

g (

in 1

mL

solu

tion)

Red

ucti

on

meth

od

Aqueo

us

dec

oct

ion

for

20

min

ute

s

Aqueo

us

dec

oct

ion

of

whole

see

ds

for

1, 5, an

d 3

0

min

ute

s an

d o

f

pow

der

ed s

eed f

or

1 a

nd 5

min

ute

s.

Aqueo

us

dec

oct

ion

for

1 h

ou

r

Aqueo

us

infu

sion

for

30

min

ute

s

Aqueo

us

dec

oct

ion

for

35

min

ute

s

Orig

inal

Safr

ole

Level

0.4

35 m

g (

in 1

g

solu

tion)

0.9

55 m

g (

in 1

g

mat

eria

l)

0.1

4 t

o 2

.76 m

g (

in 1

g m

ater

ial)

0.1

8 t

o 1

6 m

g (

in 1

g

mat

eria

l)

2.2

mg (

in 1

mL

solu

tion)

Extr

acti

on

meth

od

MeO

H

extr

acti

on

Ste

am

dis

till

atio

n

MeO

H

extr

acti

on

Aqueo

us

agit

atio

n

Pla

nt

Mate

ria

l

Cin

na

mom

u

m b

ark

Bla

ck p

epp

er

Wil

d g

ing

er

Sa

ssa

fra

s

alb

idum

-

roo

t b

ark

po

wd

er

Sa

ssa

fra

s

alb

idum

–

roo

t

Stu

dy

Rey

ner

tso

n e

t al

.,

20

05

Far

ag &

Ab

o-Z

eid

19

97

Ch

en e

t

al.,

200

9

Car

lso

n &

Th

om

pso

n 1

997

Cu

rren

t

Res

earc

h

20

12

75

Table C.2: Review of studies using laboratory animals and bacterial studies to test the

toxicity of safrole and derivatives.

Res

ult

s

No

liv

er t

um

ors

, so

me

sig

n o

f k

idn

ey

con

ges

tio

n.

At

24 m

on

ths

liv

er c

ance

rs

was

fo

un

d,

& c

ellu

lar

chan

ges

in

th

e

kid

ney

s, a

dre

nal

s, t

hy

roid

, p

itu

itar

y,

and

rep

rod

uct

ive

org

ans.

Req

uir

es p

rolo

ng

ed

exp

osu

re b

efo

re c

ellu

lar

chan

ges

.

Saf

role

was

neu

tral

or

wea

kly

po

siti

ve

Fo

r ra

ts,

wit

hin

24

ho

urs

88

% o

f th

e

low

est

do

se o

f 0

.63 m

g/k

g w

as

elim

inat

ed,

78%

of

the

60 m

g/k

g d

ose

was

eli

min

ated

, an

d 2

5%

was

eli

min

ated

at a

do

se o

f 7

45

mg

/kg.

Fo

r hu

man

s, t

he

do

se l

evel

s w

ere

abso

rbed

an

d

elim

inat

ed w

ith

in 2

4 h

ou

rs.

Un

able

to

det

ect

met

abo

lite

s in

man

.

Ex

amin

ed a

dif

fere

nt

met

abo

lic

pat

hw

ay

safr

ole

co

uld

un

der

go,

nam

ely

th

roug

h

ox

idat

ion

to

qu

ino

id m

etab

oli

tes

in l

iver

mic

roso

mes

in

a n

on

-en

zym

atic

pro

cess

.

Co

nc.

39

0 p

pm

fo

r 2

4

mo

nth

s an

d

11

70

pp

m f

or

22

mo

nth

s

Rat

s: O

ne

do

se,

fro

m 0

.63

to

75

0 m

g/k

g;

Hu

man

s: 0

.163

to 1

.655

mg

(1.6

55

mg

is

0.0

24

mg

/kg

fo

r

a 6

8 k

g p

erso

n)

Cm

pd

.

sou

rce

Nat

ura

l

oil

of

sass

afra

s

and

safr

ole

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Tri

al

Mo

del

Ro

den

t

In v

itro

Sal

mo

nel

la

Am

es t

est

Mal

e

Sp

rag

ue-

Daw

ley

rats

(10

0-

20

0 g

) an

d

hu

man

(5

0-

85

kg

)

Mal

e

Sp

rag

ue-

Daw

ley

rats

(1

80

-

20

0 g

)

Art

icle

Ab

bo

tt,

D. D

., E

.W.

Pac

km

an,

B.M

. W

agn

er a

nd

J.W

.E.

Har

riss

on

. C

hro

nic

ora

l

tox

icit

y o

f o

il o

f sa

ssaf

ras

and

safr

ole

. P

har

mac

olo

gis

t 3

: 6

2.

Bak

er,

R.S

.U.

and

A.M

.

Bo

nin

. T

ests

wit

h S

alm

on

ella

pla

te i

nco

rpo

rati

on

ass

ay.

In:

Ash

by a

nd

de

Ser

res

(ed

s.)

Ev

alu

atio

n o

f S

ho

rt-t

erm

Tes

ts f

or

Car

cin

og

ens.

Els

evie

r, N

.Y.

Ben

edet

ti,

M.

S.,

A.

Mal

on

e,

A.

L. B

roil

let.

Ab

sorp

tion

,

met

abo

lism

an

d e

xcr

etio

n o

f

safr

ole

in

th

e ra

t an

d m

an.

To

xic

olo

gy 7

(1):

69

-83

.

Bo

lto

n,

J.L

., e

t al

. E

vid

ence

that

4-a

lly

l-O

-qu

ino

nes

spo

nta

neo

usl

y r

earr

ang

e to

thei

r m

ore

ele

ctro

ph

ilic

qu

ino

ne

met

hid

es:

po

ten

tial

bio

acti

vat

ion

mec

han

ism

fo

r

the

hep

ato

carc

ino

gen

saf

role

.

Ch

em R

es.

To

xic

ol.

7:

44

3-

45

0.

Yea

r

19

61

19

85

19

77

19

94

76

Tes

tin

g t

he

theo

ry t

hat

saf

role

un

der

go

es i

n v

ivo

con

ver

sio

n t

o e

lect

rop

hy

llic

all

yli

c an

d b

enzy

lic

este

rs t

hat

are

th

e tr

ue

carc

ino

gen

s. 1

'-

hy

dro

xy

safr

ole

is

the

pri

mar

y m

etab

oli

te o

f

safr

ole

an

d a

pro

xim

ate

carc

ino

gen

, m

ore

so

th

an

safr

ole

.

Sh

ow

ed 1

'-h

yd

roxy

safr

ole

is

mo

re c

arci

no

gen

ic

than

saf

role

in

ad

ult

mal

e ra

ts,

alm

ost

hal

f o

f ra

ts

giv

en 1

'-h

yd

rox

ysa

fro

le d

evel

op

ed l

iver

carc

ino

mas

by

8 m

on

ths

ver

sus

rats

giv

en s

afro

le

0.0

4%

dev

elo

pin

g l

iver

car

cin

om

as b

y 1

6

mo

nth

s. T

he

rod

ents

gai

ned

wei

gh

t m

ore

slo

wly

,

bu

t un

til

they

sta

rted

dev

elo

pin

g t

um

ors

,

surv

ivo

rsh

ip w

as h

igh

.

Saf

role

tes

ted

po

siti

ve

for

Sis

ter

Ch

rom

atid

Ex

chan

ge

assa

y.

Sh

ow

ed t

hat

man

y o

f th

e co

mpo

un

ds

vo

lati

lize

d

du

rin

g t

he

incu

bat

ion

per

iod.

Tes

ted

th

e re

cov

ery

of

the

com

po

un

ds

fro

m h

ou

sefl

ies

and

mic

e.

90

mg

was

inje

cted

in

to

rats

or

30

mg

/100

g b

ody

wei

gh

t

Rat

s an

d m

ice:

fed

via

die

t

con

c. o

f 0

.5%

,

0.4

%,

or

0.3

%

safr

ole

, 0

.55

%

or

0.4

4%

1'-

hy

dro

xy

safr

ole

for

8.5

to

11

mo

nth

s

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Sta

nd

ard

Rat

s (3

00

g),

ham

ster

s,

and

gu

inea

pig

s.

CD

rat

s

(23

0-2

60

g)

and

CD

-

1 m

ice

(27

-

29

g)

In v

ivo

mam

mal

ia

n c

ell

gen

oto

xic

it

y a

ssay

Ho

use

fly

and

mal

e

alb

ino

mic

e

Bo

rch

ert,

P.,

et

al.

Th

e

met

abo

lism

of

the

nat

ura

lly

occ

urr

ing

hep

ato

carc

ino

gen

safr

ole

to

1'-

hyd

rox

ysa

fro

le

and

th

e el

ectr

op

hil

ic

reac

tiv

ity

of

1'-

acet

ox

ysa

fro

le.

Can

cer

Res

earc

h 3

3:

57

5-5

89.

Bo

rch

est,

P.,

et

al.

1'-

hy

dro

xy

safr

ole

, a

pro

xim

ate

carc

ino

gen

ic m

etab

oli

te o

f

safr

ole

in

th

e ra

t an

d m

ou

se.

Can

cer

Res

. 3

3:

590

- 60

0.

Bra

dle

y,

M.O

. M

easu

rem

ent

of

DN

A s

ing

le-s

tran

d b

reak

s

by

alk

alin

e el

uti

on

in

rat

hep

ato

cyte

s. I

n:

Ash

by a

nd

de

Ser

res

(ed

s).

Ev

alu

atio

n

of

Sho

rt-t

erm

Tes

ts f

or

Car

cin

og

ens.

Els

evie

r, N

.Y.,

35

3-3

57

.

Cas

ida,

J.

E.,

et

al.

Met

hy

len

e-C

14-d

iox

yp

hen

yl

com

pou

nd

s:

Met

abo

lism

in

rela

tio

n t

o t

hei

r sy

ner

gis

tic

acti

on

. S

cien

ce 1

53

(37

40

):

11

30

-11

33

19

73

19

73

19

85

19

66

77

Saf

role

an

d s

ix d

eriv

ativ

es w

ere

test

ed-

the

der

ivat

ives

pro

ved

to

be

mo

re c

yto

tox

ic t

o

hu

man

can

cer

cell

s th

an s

afro

le.

Rat

s w

ere

use

d t

o e

stab

lish

a l

inea

r d

ose

-

resp

on

se r

elat

ion

ship

- fo

und

th

at t

her

e w

as a

met

abo

lic

satu

rati

on

bet

wee

n 1

50 a

nd

300

mg

/kg i

n r

ats.

Th

e sa

fro

le m

etab

oli

tes

dih

yd

roxy

chav

ico

l an

d e

ug

eno

l w

ere

fou

nd

in t

he

uri

ne

of

non

-bet

el q

uid

ch

ewer

s, a

t th

e

lev

els

of

no

t d

etec

ted

to

5.4

mic

rog

ram

s/m

g

crea

tin

ine,

pro

bab

ly d

ue

to s

pic

es i

n t

he

die

t.

Liv

er w

eig

ht

incr

ease

d t

o 2

0%

aft

er w

eek

1.

Liv

er w

eig

ht

con

tinu

ed t

o i

ncr

ease

to

60

%

mo

re t

han

th

e co

ntr

ol

gro

up

by w

eek

8 w

ith

a d

ecre

ase

in d

rug

met

abo

lizi

ng

en

zym

e

acti

vit

y.

Cy

toch

rom

e P

-450

was

in

itia

lly

80

% h

igh

er,

bu

t lo

wer

ed t

o 2

0-3

0%

hig

her

afte

r 1

6 w

eek

s. N

oti

ced

en

larg

ed

cen

tril

ob

ula

r h

epat

ocy

tes,

in

div

idu

al c

ell

nec

rosi

s, a

nd

nec

rosi

s o

f h

epat

ocy

tes.

Use

d 3

2P

-po

stla

bel

ing

ass

ay t

hat

saf

role

form

s D

NA

add

uct

s, i

n t

urn

in

du

ces

sist

er

chro

mat

id e

xch

ang

es a

nd

ch

rom

oso

mal

aber

rati

on

s as

say

s.

Rat

s: 0

, 3

0, 75

,

15

0,

30

0 m

g/k

g.

Tes

ted

uri

ne

of

rats

an

d h

um

an

wh

o c

hew

ed

qu

id b

etw

een

2

- 1

00 t

imes

a

day

Die

t co

nta

inin

g

0.2

5%

(w

/w)

safr

ole

fo

r 8

5

wee

ks,

10

%

mo

re s

afro

le

was

in

itia

lly

add

ed d

ue

to

vo

lati

lity

0.0

25

to

0.2

mg

/ml

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Bre

ast

and

colo

rect

al

can

cer

cell

lin

es

Ad

ult

mal

e

Wis

tar

rats

an

d

hu

man

uri

ne

Fem

ale

Wis

tar

alb

ino

rat

s

(80

-100

g)

Ch

ines

e

ham

ster

lun

g c

ells

Cat

alán

, L

. E

., e

t al

. S

yn

thes

is o

f

Nin

e S

afro

le D

eriv

ativ

es a

nd

Th

eir

An

tip

roli

fera

tiv

e A

ctiv

ity

To

war

ds

Hu

man

Can

cer

Cel

ls.

Jou

rnal

of

the

Ch

ilea

n C

hem

ical

So

ciet

y 5

5:

2, 2

19

-222

.

Ch

ang,

M.J

.W., C

.Y. K

o, R

.F.

Lin

& L

.L.

Hsi

eh.

Bio

log

ical

mo

nit

ori

ng

of

env

iro

nm

ent

exp

osu

re t

o s

afro

le a

nd

th

e

Tai

wan

ese

bet

el q

uid

ch

ewin

g.

Arc

h.

Env

iro

n.

Co

nta

m. T

ox

ico

l.

43

: 43

2–

43

7.

Cra

mp

ton

, R

.F.,

et

al.

Lo

ng

-ter

m

stu

die

s o

n c

hem

ical

ly i

nd

uce

d

liv

er e

nla

rgem

ent

in t

he

rat.

II.

Tra

nsi

ent

ind

uct

ion

of

mic

roso

mal

en

zym

es l

ead

ing

to

liv

er d

amag

e an

d

no

du

larh

yp

erp

lasi

a p

rod

uce

d b

y

safr

ole

an

d P

on

ceau

MX

.

To

xic

olo

gy 7

: 3

07

-326

.

Dai

mo

n H

., S

. S

awad

a, S

.

Asa

ku

ra &

F. S

agam

i. A

nal

ysi

s

of

cyto

gen

etic

eff

ects

an

d D

NA

add

uct

fo

rmat

ion

ind

uce

d b

y

safr

ole

in

Ch

ines

e h

amst

er l

un

g

cell

s. T

erat

og

enes

is

Car

cin

og

enes

is M

uta

gen

esis

17

(1):

7-1

8.

20

10

20

02

19

77

19

97

78

No

mo

rtal

ity

. T

he

sin

gle

do

ses

did

no

t in

du

ce

chro

mo

som

e ab

erra

tio

n,

bu

t th

e re

pea

ted

do

ses

incr

ease

d a

ber

ran

t ce

lls

in t

he

liv

er.

Th

e

sin

gle

do

ses

at 1

00

, 2

50

and

500

mg

/kg

an

d

the

rep

eate

d d

ose

s in

du

ced

sis

ter

chro

mat

id

exch

ang

es.

DN

A a

ddu

cts

wer

e fo

un

d i

n t

he

sin

gle

do

ses

of

10 (

2 a

dd

uct

s) a

nd

10

0,

25

0,

& 5

00

(4 a

ddu

cts)

mg

/kg

wh

ile

no a

ddu

cts

wer

e o

bse

rved

in

1 m

g/k

g

Pu

rpo

se o

f th

is s

tud

y w

as t

o d

isco

ver

th

e

acti

vat

ion

or

enh

ance

men

t o

f sa

fro

le t

hat

tes

ts

po

siti

ves

fo

r th

e A

mes

tes

t. S

afro

le t

este

d

po

siti

ve

usi

ng

str

ain

TA

153

5, ac

tiv

ated

by

rat

liv

er i

nje

cted

wit

h s

afro

le i

n v

ivo

. P

rev

iou

s

stu

die

s te

sted

saf

role

wit

ho

ut

bei

ng

mod

ifie

d

and

sh

ow

ed s

afro

le t

o b

e n

egat

ive

in a

n A

mes

test

.

Th

e 1

'-h

yd

roxy

met

abo

lite

s o

f b

oth

est

rag

ole

and

saf

role

wer

e fo

un

d t

o b

e m

ore

carc

ino

gen

ic t

han

th

e p

aren

t co

mp

ou

nd

s. 5

9%

of

the

mic

e g

iven

1'-

hyd

roxy

safr

ole

dev

elo

ped

liv

er t

um

ors

.

Fiv

e d

ose

s o

f

62

.5, 1

25

or

25

0 m

g/k

g,

sin

gle

do

ses

of

1,

10

, 1

00

, 2

50

,

or

500

mg

/kg

via

sto

mac

h

tub

e

42

mg

/rat

/day

via

i.p

.

inje

ctio

n f

or

thre

e d

ays

New

bo

rn m

ice:

tota

l o

f 4

.43

mic

rom

ole

s; 9

-

12

wee

k o

ld

mic

e: t

ota

l o

f

5.1

9

mic

rom

ole

s,

via

se

inje

ctio

n

Saf

role

Sta

nd

ard

Saf

role

stan

dar

d,

syn

thes

iz

ed t

o 1

'-

hy

dro

xy

safr

ole

Rat

(F3

44

)

mo

del

Ad

ult

rats

CD

-1

mic

e,

new

bo

rn

(16

g)

and

9-1

2

wee

k o

ld

(35

g)

Dai

mo

n, H

., S

. S

awad

a, S

.

Asa

ku

ra,

and

F.

Sag

ani.

In

viv

o

gen

oto

xic

ity

an

d D

NA

add

uct

lev

els

in t

he

liv

er o

f ra

ts t

reat

ed

wit

h s

afro

le.

Car

cin

og

enes

is 1

9:

14

1-1

46

.

Do

ran

ge,

J. L

. et

al.

Co

mp

arat

ive

surv

ey o

f m

icro

som

al a

ctiv

atio

n

syst

ems

for

mu

tag

enic

stu

die

s o

f

safr

ole

, M

uta

tio

n R

esea

rch

5

3:

17

9-1

80

.

Dri

nk

wat

er,

N.

R. et

al.

Hep

ato

carc

ino

gen

icit

y o

f es

trag

ole

(1 a

lly

l-4

-met

ho

xyb

enze

ne)

an

d

1'-

hyd

roxy

estr

ago

le i

n t

he

mo

use

and

mu

tag

enic

ity

of

1'-

acet

ox

yes

trag

ole

in

bac

teri

a. J

.

Nat

l. C

ance

r In

st.

57

: 1

32

3-1

331

.

19

98

19

78

19

76

79

S

afro

le r

eact

s to

fo

rm a

saf

role

met

abo

lite

- cy

toch

rom

e P

-45

0

com

ple

x.

Do

min

ant

leth

al a

ssay

: sa

fro

le t

este

d

neg

ativ

e

Th

e b

ile

and

uri

ne

sam

ple

s sh

ow

ed

safr

ole

met

abo

lize

d i

nto

iso

safr

ole

and

dih

yd

rosa

fro

le.

Th

e sa

fro

le a

nd

met

abo

lite

s w

ere

slo

wly

eli

min

ated

in

the

bil

e.

2%

(w

/w)

via

die

t fo

r tw

o

wee

ks.

0.0

4 m

l v

ia

intr

aven

ou

s

inje

ctio

n

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Mal

e

Sp

rag

ue-

Daw

ley

rats

(2

00

g)

Sw

iss

mic

e 8

-10

wee

ks

old

Sp

rag

ue-

Daw

ley

mic

e (3

50

g)

Elc

om

be,

C.

R. et

al.

Stu

die

s o

n

the

inte

ract

ion

of

safr

ole

wit

h r

at

hep

atic

mic

roso

mes

. B

ioch

em.

Ph

arm

aco

l. 2

4:

142

7-1

433

.

Ep

stei

n,

S.S

., e

t al

. D

etec

tio

n o

f

chem

ical

mu

tag

ens

by

th

e

do

min

ant

leth

al a

ssay

in

th

e

mo

use

. T

ox

ico

log

y a

nd

App

lied

Ph

arm

aco

log

y 2

3:

28

8-

32

5.

Fis

hb

ein

, L

. et

al.

Th

in-l

ayer

chro

mat

og

rap

hy

of

rat

bil

e an

d

uri

ne

foll

ow

ing i

ntr

aven

ou

s

adm

inis

trat

ion

of

safr

ole

,

iso

safr

ole

, an

d d

ihy

dro

safr

ole

. J.

Ch

rom

atog

. 2

9:

26

7-2

73

.

19

75

19

72

19

67

80

Th

e sa

fro

le h

ad n

o e

ffec

t o

n

mic

roso

mal

en

zym

e ac

tiv

ity

.

Saf

role

tes

ted

neg

ativ

e

Saf

role

was

neu

tral

or

wea

kly

po

siti

ve

Ex

amin

ed D

NA

usi

ng

32P

-po

stla

bel

ing

assa

y a

t 0

.5, 1

, 2

, 3

, 7

, 1

5,

and

30

day

s.

Sh

ow

ed a

lin

ear

resp

on

se o

f sa

fro

le-

DN

A a

dd

uct

s, r

each

ing

a t

hre

sho

ld a

t

the

10

mg

do

se,

wh

ere

the

tiss

ue

cou

ld

be

satu

rate

d a

nd

th

e el

imin

atio

n o

f th

e

com

pou

nd

wo

uld

occ

ur

mo

re s

low

ly.

10

mg

/kg

on

e

tim

e d

ose

of

safr

ole

via

i.p

.

inje

ctio

n

On

e do

se o

f

0.0

01

, 0

.01,

0.1

,

1.0

, an

d 1

0.0

mg

/mou

se o

r

0.0

4, 0

.4,

4,

40

,

or

400

mg

/kg

bw

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Mal

e

Sw

iss

Alb

ino

mic

e (2

0-

25

g)

Bo

ne-

mar

row

mic

ron

ucl

eus

assa

y

In v

itro

Sal

mo

nel

l

a re

ver

se

mu

tati

on

assa

y

(Am

es

test

)

Fem

ale

CD

-1

mic

e (2

5

g)

Fri

edm

an,

M. A

., E

. A

rno

ld,

Y.

Bis

ho

p, an

d S

.S.

Ep

stei

n.

Ad

dit

ive

and s

yn

erg

isti

c

inh

ibit

ion

of

mam

mal

ian

mic

roso

mal

en

zym

e fu

nct

ion

s

by

pip

ero

ny

l bu

tox

ide,

saf

role

and

oth

er m

eth

yle

ned

ioxy

ph

eny

l

der

ivat

ives

. E

xp

erie

nti

a 2

7:

10

52

-10

54

.

Go

cke,

E.,

M.T

. K

ing,

K.

Eck

ard

t, a

nd

D.W

ild

.

Mu

tag

enic

ity

of

cosm

etic

s

ing

red

ien

ts l

icen

sed

by

th

e

Eu

rop

ean

Com

mun

ity

. M

uta

t.

Res

. 9

0:

91

-10

9.

Gre

en,

N.R

. an

d S

avag

e, J

.R.,.

Scr

een

ing

of

safr

ole

, eu

gen

ol,

thei

r n

inh

yd

rin

po

siti

ve

met

abo

lite

s an

d s

elec

ted

seco

nd

ary

am

ines

fo

r p

ote

nti

al

mu

tag

enic

ity

. M

uta

tio

n

Res

earc

h,

57

, 1

15

- 12

1.

Gu

pta

, K

.P., K

.L. v

an G

ole

n,

K.L

. P

utm

an &

K.

Ran

der

ath

.

Fo

rmat

ion

and

per

sist

ence

of

safr

ole

-DN

A a

dd

uct

s o

ver

a

10

,000

-fo

ld d

ose

ran

ge

in m

ou

se

liv

er.

Car

cin

og

enes

is 1

4 (

8):

15

17

–1

52

1.

19

71

19

81

19

78

19

93

81

T

he

rats

fee

d 1

0,0

00

pp

m d

ied

aft

er

62

wee

ks,

and

th

e re

st e

xp

erie

nce

d g

row

th r

etar

dat

ion

,

incr

ease

d m

ort

alit

y i

n t

he

mal

es (

at 5

00

0pp

m),

liv

er e

nla

rgem

ent

wit

h t

um

or

mas

ses

and

no

du

les,

mil

d h

yp

erp

lasi

a o

f th

yro

id, an

d

incr

ease

of

chro

nic

nep

hri

tis

in t

he

kid

ney

. T

he

do

gs

exp

erie

nce

d l

iver

en

larg

emen

t an

d n

odu

les

at t

he

20

mg

/kg d

ose

an

d l

iver

dam

age

by

fo

cal

nec

rosi

s, b

ile-d

uct

pro

life

rati

on

, fa

tty

met

amo

rph

osi

s, a

nd

oth

er c

han

ges

at

the

5

mg

/kg d

ose

.

At

750

mg

/kg,

9 o

ut

of

10

rat

s d

ied

at

19 d

ays,

at 5

00

mg

/kg

, 1

of

10 r

ats

die

d a

fter

46

day

s,

and

at

250

mg

/kg

, n

o r

ats

die

d b

ut

they

exp

erie

nce

d l

iver

en

larg

emen

t an

d f

oca

l

nec

rosi

s. N

o e

vid

ence

of

tox

icit

y f

or

iso

safr

ole

.

Dih

yd

rosa

fro

le w

as f

ou

nd

to

be

a es

op

hag

eal

carc

ino

gen

. M

ice

sho

wed

sim

ilar

liv

er c

han

ges

as f

ou

nd

in

th

e ra

ts.

Th

e ra

ts r

ecei

vin

g h

igh p

rote

in d

iets

(3

0%

pro

tein

), t

he

liv

er a

pp

eare

d n

orm

al c

om

par

ed t

o

the

con

tro

l w

hil

e o

ther

die

ts w

ith

lo

wer

pro

tein

had

sm

alle

r li

ver

s w

hen

fed

wit

h s

afro

le.

Th

e

safr

ole

sp

iked

die

ts r

esu

lted

in

liv

ers

wit

h n

o f

at

pre

sen

t. R

ats

giv

en s

afro

le l

ived

lo

ng

er t

han

th

e

con

tro

l, b

ut

ther

efo

re h

ad a

hig

her

lev

el o

f

hep

ato

mas

.

Rat

s, a

du

lt:

10

00

, 2

50

0,

50

00

, an

d

10

,000

pp

m

for

two

yea

rs;

Do

gs

(2M

,

2F

): 5

and

20

mg

/kg f

or

6

yea

rs

Rat

s, a

du

lt:

25

0,

50

0,

75

0

mg

/kg

/day

for

105

day

s;

Mic

e: 2

50

and

50

0

mg

/kg f

or

60

day

s

0.5

% s

afro

le

via

die

t fo

r

fiv

e d

iets

wit

h v

ary

ing

lev

els

of

pro

tein

, fa

t,

carb

oh

yd

rate

s, a

nd

sal

ts.

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Osb

ou

rne-

Men

del

rats

an

d

pu

re-

bre

ed

bea

gle

do

gs

Osb

ou

rne-

Men

del

rats

an

d

Sw

iss

mic

e

Osb

ou

rne-

Men

del

mal

e ra

ts

(11

4 g

)

Hag

an,

E.C

., W

.H.

Han

sen

, O

.G.

Fit

zhug

h,

P.M

. Je

nn

er,

W.I

. Jo

nes

,

J.M

, T

aylo

r, E

.L.

Lo

ng

,

A.A

. N

elso

n,

and

J.B

.

Bro

uw

er.

Fo

od

flav

ou

rin

gs

and

com

pou

nd

s o

f re

late

d

stru

ctu

re.

II.

Su

bac

ute

an

d

chro

nic

to

xic

ity

. F

ood

Co

sm. T

ox

ico

l. 5

: 1

41

-

15

7.

Hag

an,

E.C

., P

. M

. Je

nn

er,

W.

I. J

on

es,

O.

G.

Fit

zhu

gh,

E.

L. L

ong

, J.

G.

Bro

uw

er,

W. W

elfa

re.

To

xic

pro

per

ties

of

com

pou

nd

s re

late

d t

o

safr

ole

. T

ox

ico

log

y a

nd

Ap

pli

ed P

har

mac

olo

gy

7(1

): 1

8-2

4.

Ho

mbu

rger

, F

., P

.D.

Bo

gd

on

off

and

T.F

.

Kel

ley

. In

flu

ence

of

die

t

on

ch

ron

ic o

ral

tox

icit

y o

f

safr

ole

an

d b

utt

er y

ello

w

in r

ats.

P

roce

edin

gs

of

the

So

ciet

y f

or

Exp

erim

enta

l

Bio

logy

and

Med

icin

e 1

19

(4):

11

06

-11

10

.

19

67

19

65

19

65

82

S

afro

le t

este

d p

osi

tiv

e fo

r

gen

oto

xic

ity

act

ivit

y,

at a

hig

her

do

se

than

pre

vio

us

stu

die

s (P

rob

st e

t al

.

19

81

) th

at t

este

d s

mal

l d

ose

s.

Bo

th i

nse

cts

sho

wed

su

scep

tib

ilit

y t

o

the

fum

igan

t to

xic

ity

of

safr

ole

. T

her

e

was

sli

gh

t fe

edin

g d

eter

ren

ce a

gai

nst

S.

zea

ma

is d

ue

to r

edu

ced

fo

od

con

sum

pti

on

and

no

det

erre

nce

fo

r T

.

cast

an

eum

.

Saf

role

did

no

t ef

fect

gro

wth

of

A.

act

ino

myc

etem

com

ita

ns

or

S. m

uta

ns,

bu

t d

id d

ecre

ase

the

gro

wth

of

E. co

li.

Saf

role

red

uce

s th

e re

leas

e o

f

sup

ero

xid

e an

ion

, b

ut

no

t th

roug

h a

dir

ect

kil

lin

g o

f n

eutr

oph

ils,

or

a h

ost

def

ense

mec

han

ism

. S

afro

le r

edu

ced

anti

mic

rob

ial

acti

vit

y b

ut

did

no

t

dem

on

stra

te c

yto

tox

icit

y.

10

^-3

M s

afro

le

for

a

un

sch

edu

led

DN

A s

yn

thes

is

assa

ys

in

cult

ure

d r

at

hep

ato

cyte

s.

Fu

mig

ant

stud

y

at 0

.9 m

g/c

m^3

and

co

nta

ct

tox

icit

y s

tud

y a

t

2.0

4-1

6.2

mg

/g

of

food

.

5 m

M a

nd

10

mM

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Mal

e F

isch

er

34

4 r

ats.

Sit

op

hil

us

zea

ma

is a

du

lts

and

Tri

bo

liu

m

cast

an

eum

adu

lts

and

larv

ae

Ora

l

pat

ho

gen

s

incl

ud

ing

Act

ino

ba

cill

us

act

ino

myc

etem

com

ita

ns,

Str

epto

cocc

us

mu

tan

s,

and

Po

ryp

hyr

om

on

as

gin

giv

ali

s

Ho

wes

, J.

A., V

.S.W

. C

han

,

and

J.

Cal

dw

ell.

Str

uct

ure

-

spec

ific

ity

of

the

gen

oto

xic

ity

of

som

e n

atu

rall

y o

ccu

rrin

g

alk

eny

l-b

enze

nes

det

erm

ined

by

th

e u

nsc

hed

ule

d D

NA

syn

thes

is a

ssay

s in

rat

hep

ato

cyte

s. F

ood

Ch

em.

To

xic

ol.

28

: 5

37

-54

2.

Hu

ang,

Y. H

o S

H,

Kin

i R

M.

Bio

acti

vit

ies

of

safr

ole

an

d

iso

safr

ole

on

Sit

op

hil

us

zea

ma

is (

Co

leo

pte

ra:

Cu

rcu

lio

nid

ae)

and

Tri

bo

liu

m

cast

an

eum

(C

ole

op

tera

:

Ten

ebri

on

idae

). J

ou

rnal

of

Eco

nom

ic E

nto

mo

log

y 9

2:

67

6-6

83

.

Hu

ng

, S

.-L

., Y

.-L

. C

hen

, an

d

Y.-

T.

Ch

en. E

ffec

ts o

f sa

fro

le

on

th

e d

efen

siv

e fu

nct

ion

s o

f

hu

man

neu

trop

hil

s. J

ou

rnal

of

Per

iod

on

tal

Res

earc

h 3

8:

130

–

13

4.

19

90

19

99

20

03

83

Liv

er-c

ell

tum

ors

fo

und

in

14 o

f

34

mal

es a

nd

32

of

33

fem

ales

.

Saf

role

tes

ted

po

siti

ve

Est

abli

shed

acu

te t

ox

icit

y a

t

thes

e co

nce

ntr

atio

ns.

Hy

bri

d m

ice,

7

day

s o

ld:

464

mg

/kg o

ver

21

day

s v

ia

sto

mac

h t

ub

e,

then

12

65

mg

/kg o

ver

82

wee

ks

via

die

t

19

50

mg

/kg

bw

for

rats

an

d

23

50

mg

/kg

fo

r

mic

e

Saf

role

stan

dar

d

Hy

bri

d m

ice

fro

m C

57

BL

/6

(fem

ale)

an

d

C3

H/A

nf

or

AK

R (

mal

e)

stra

ins

In v

ivo

mam

mal

ian

cell

gen

oto

xic

ity

assa

y:

chro

mo

som

al

aber

rati

on

s

Rat

an

d m

ice

Inn

es,

J.R

., B

.M. U

llan

d,

M.G

. V

aler

io,

L.

Pet

ruce

lli,

L.

Fis

hb

ein

, E

.R.

Has

t,

A.J

. P

allo

ta,

R.R

. B

ates

, H

.L. F

alk

, L

.L.

Gar

t, M

. K

lein

, I.

Mit

chel

l, a

nd

J.

Pet

er.

Bio

assa

y o

f p

esti

cid

es a

nd

in

du

stri

al

chem

ical

s fo

r tu

mo

rig

enic

ity

in

mic

e: a

pre

lim

inar

y n

ote

. J.

Nat

l. C

ance

r In

st.

42

: 11

01

-11

14

.

Ish

idat

e, M

. an

d T

. S

ofu

ni.

Th

e in

vit

ro

chro

mo

som

al a

ber

rati

on

tes

t u

sin

g

Ch

ines

e h

amst

er l

un

g (

CH

L)

fib

rob

last

cell

s in

cu

ltu

re.

In:

Ash

by a

nd

de

Ser

res

(ed

s).

Ev

alu

atio

n o

f S

ho

rt-t

erm

Tes

ts

for

carc

ino

gen

s. E

lsev

ier,

N.Y

.: 4

27

-

43

2.

Jen

ner

, P

.M.,

E.C

. H

agan

, J.

M. T

aylo

r,

E.L

. C

oo

k,

and O

.G.

Fit

zhug

h.

Fo

od

flav

ou

rin

gs

and c

om

po

un

ds

of

rela

ted

stru

ctu

res.

I.

Acu

te O

ral

To

xic

ity

. F

oo

d

Co

sm. T

ox

ico

l. 2

: 3

27

-343

.

19

69

19

85

19

64

84

Fo

un

d t

he

form

atio

n o

f 1

'-hy

dro

xy

safr

ole

in t

he

hu

man

liv

er m

icro

som

es.

Var

iou

s

hu

man

cy

toch

rom

e P

45

0 e

nzy

mes

met

abo

lize

saf

role

, in

clu

din

g e

nzy

mes

P4

50

2C

9*

1,

P4

50

2A

6,

P4

50

2D

6*

1,

and

P4

50

2E

1.

Bo

th d

ose

s ex

per

ien

ced

dec

reas

ed b

od

y

wei

gh

t g

ain

, li

ver

en

larg

emen

t in

th

e m

ales

and

fem

ales

(0

.5%

gro

up

on

ly),

an

d s

ing

le

cell

nec

rosi

s. M

ale

rats

ex

per

ien

ced

tub

ula

r h

yal

ine

dro

ple

ts,

tub

ula

r

reg

ener

atio

n,

gra

nu

lar

cast

, p

elv

ic

calc

ific

atio

n,

and i

nte

rsti

tial

cel

l

infi

ltra

tio

n i

n t

he

kid

ney

s.

Th

e m

ajo

r m

etab

oli

c p

ath

way

fo

r sa

fro

le i

s

clea

vag

e o

f th

e m

eth

yle

ned

iox

yp

hen

yl

mo

iety

as

wel

l as

ex

pir

atio

n o

f ca

rbo

n

dio

xid

e fr

om

th

e m

eth

yle

ne

carb

on

.

Met

abo

lism

of

safr

ole

, (w

hic

h i

s v

ola

tile

)

resu

lted

in

les

s v

ola

tile

, m

ore

po

lar,

an

d

eth

er-s

olu

ble

met

abo

lite

s. F

ou

nd

th

e

met

abo

lite

s o

f sa

fro

le w

ere

mo

re v

ola

tile

.

Th

e sa

fro

le-f

ree

sass

afra

s ex

trac

t p

rod

uce

d

loca

l tu

mo

rs i

n 6

6%

of

the

rats

, th

ou

gh

stu

dy

do

es n

ot

ind

icat

ed i

f th

e sa

mp

les

wer

e te

sted

fo

r sa

fro

le l

evel

s p

rio

r to

inje

ctio

n.

Rat

s: 6

9.1

(0.1

% i

n

die

t) a

nd

27

5.6

(0.5

%)

mg

/kg

/day

for

13

wee

ks

Mic

e: 5

mic

rom

ole

s/k

g b

w;

rats

&

ham

ster

s:

10

mic

rom

ole

s/k

g b

w

Rat

s: 1

5

mg

via

s.c

.

inje

ctio

n

for

78

wee

ks

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Sas

safr

as r

oo

t

bar

k,

extr

acte

d w

ith

pet

role

um

eth

er,

met

hy

len

e

chlo

rid

e, a

nd

eth

ano

l

Hu

man

cyto

chro

me

P4

50

en

zym

es

and

mic

roso

mes

fro

m l

iver

cell

s li

nes

Mal

e an

d

Fem

ale

F3

44

gp

t d

elta

rat

s

Mal

e S

wis

s-

Web

ster

mic

e

(18

-20 g

),

mal

e S

pra

gu

e-

Daw

ley

rat

s

(15

0-1

70 g

),

mal

e h

amst

er

(18

0-2

00 g

)

NIH

Bla

ck

rats

, 1

-2

mo

nth

old

Jeu

riss

en,

S.

M. F

. et

al.

Hum

an

Cy

toch

rom

e P

45

0 E

nzy

me

Sp

ecif

icit

y f

or

Bio

acti

vat

ion

of

Saf

role

to

th

e P

rox

imat

e

Car

cin

og

en 1

-Hy

dro

xy

safr

ole

.

Ch

emic

al R

esea

rch

in

To

xic

olo

gy.

17

(9

): 1

24

5-1

250

.

Jin

, M

., A

. K

ijm

a, Y

. S

uzu

ki,

D.

Hib

i, T

. In

ou

e, Y

. Is

hii

, T

.

No

hm

i, A

. N

ish

ikaw

a, K

.

Og

awa,

T. U

men

ura

.

Co

mp

reh

ensi

ve

tox

icit

y s

tud

y o

f

safr

ole

usi

ng

a m

ediu

m-t

erm

anim

al m

od

el w

ith

gp

t d

elta

rat

s.

To

xic

olo

gy 2

90

: 3

12

-321

.

Kam

ien

ski,

F.X

. an

d J

.E.

Cas

ida.

Im

po

rtan

ce o

f

dem

eth

yle

nat

ion

in

th

e

met

abo

lism

in

viv

o a

nd

in

vit

ro

of

met

hy

len

e-d

ioxy

ph

eny

l

syn

erg

ists

an

d r

elat

ed

com

pou

nd

s in

mam

mal

s.

Bio

chem

ical

Ph

arm

aco

lolg

y 1

9:

91

-11

2.

Kap

adia

, G

. J.

, E

. B

. C

hun

g,

B.

Gh

osh

, Y

. N

. S

hu

kla

, S

. P

.

Bas

ak,

J. F

. M

ort

on

, S

. N

.

Pra

dh

an. C

arci

no

gen

icit

y o

f

som

e fo

lk m

edic

inal

her

bs

in

rats

. Jo

urn

al o

f N

atio

nal

Can

cer

Inst

itu

te 6

0 (

3):

683

-686

.

20

04

20

11

19

70

19

78

85

B.

sub

tili

s w

as n

ot

inh

ibit

ed b

y

safr

ole

or

safr

ole

ep

ox

ide,

bu

t it

was

inh

ibit

ed b

y s

afro

le h

yd

rop

ero

xid

e.

S.

au

reu

s w

as a

lso

no

t in

hib

ited

by

safr

ole

, b

ut

it w

as b

y s

afro

le e

po

xid

e

and

saf

role

hy

dro

per

ox

ide.

E.

coli

was

in

hib

ited

by a

ll t

hre

e

com

pou

nd

s.

Th

e ph

oto

syn

thes

ized

co

mp

ou

nd

s

der

ived

fro

m s

afro

le i

nh

ibit

ed

gro

wth

of

Ca

nd

ida a

lbic

an

s.

Saf

role

was

eff

ecti

ve

agai

nst

th

e ri

ce

wee

vil

via

fu

mig

atio

n,

lik

ely

th

rou

gh

vap

or

acti

on

th

rou

gh

th

e re

spir

ato

ry

syst

em.

Fo

un

d t

hat

th

e es

sen

tial

oil

dir

ectl

y f

rom

th

e p

lan

t w

as m

ore

effe

ctiv

e th

an p

uri

fied

co

mp

oun

ds,

as t

he

com

po

un

ds

in t

he

esse

nti

al o

il

fro

m t

he

pla

nt

cou

ld b

e ac

tin

g

syn

erg

isti

call

y.

25

mic

rog

ram

/m

L o

f es

sen

tial

oil

on

fil

ter

pap

er f

or

the

fum

igan

t

bio

assa

y

Saf

role

stan

dar

d

Saf

role

stan

dar

d,

ph

oto

syn

th

esiz

ed t

o

com

pou

nd

Asi

asa

rum

sieb

old

i

esse

nti

al

oil

Gra

m-p

osi

tiv

e

bac

teri

a B

aci

llu

s

sub

till

is,

Sta

ph

ylo

cocc

us

au

reu

s, a

nd

gra

m-n

egat

ive

bac

teri

a E

. co

li

Ca

nd

ida

alb

ica

ns,

fun

gal

pat

ho

gen

Fu

mig

atio

n

bio

assa

y a

gai

nst

the

rice

wee

vil

Sit

op

hil

us

ory

zae

Kh

ayy

at, S

. A

an

d S

. H

. A

l-Z

ahra

ni.

Th

erm

al,

pho

tosy

nth

esis

an

d

anti

bac

teri

al s

tud

ies

of

bio

acti

ve

safr

ole

der

ivat

ive

as p

recu

rso

r fo

r

nat

ura

l fl

avo

r an

d f

rag

ran

ce.

Ara

bia

n J

ou

rnal

of

Ch

emis

try

,

do

i:1

0.1

016

/j.a

rab

jc.2

01

1.0

9.0

14.

Kh

ayy

at, S

. A

. P

ho

tosy

nth

esis

of

dim

eric

cin

nam

ald

ehy

de,

eu

gen

ol,

and

saf

role

as

anti

mic

rob

ial

agen

ts.

Jou

rnal

of

Sau

di

Ch

emic

al S

oci

ety

,

do

i: 1

0.1

016

/j.j

scs.

20

11.0

7.0

14.

Kim

, J.

an

d I

-K.

Par

k. F

um

igan

t

tox

icit

y o

f K

ore

an m

edic

inal

pla

nt

esse

nti

al o

ils

and

co

mpo

nen

ts f

rom

Asi

asa

rum

sie

bo

ldi

roo

t ag

ain

st

Sit

op

hil

us

ory

zae

L.

Fla

vou

r an

d

Fra

gra

nce

Jo

urn

al 2

3:

79

-83.

20

11

20

11

20

08

86

Ind

uct

ion

of

ben

zopy

ren

e h

yd

roxy

lase

, w

ith

max

imu

m e

nzy

me

acti

vit

y w

ith

in 1

4 d

ays.

Incr

ease

s w

her

e o

bse

rved

in

hep

atic

bip

hen

yl

2-

hy

dro

xy

lase

an

d 4

-hyd

rox

yla

se a

ctiv

ity

,

mic

roso

mal

pro

tein

, li

ver

wei

gh

t, c

yto

chro

me

P-4

50

co

nte

nt.

Hep

atic

an

ilin

e 4

-hy

dro

xy

lase

acti

vit

y w

as i

nh

ibit

ed.

Saf

role

in

du

ces

a d

ose

-dep

end

ent

incr

ease

of

hep

atic

lip

id h

yd

rop

ero

xid

es a

nd

8-h

yd

roxy

-2'-

deo

xy

gu

ano

sin

e. T

he

hig

her

th

e do

se,

the

earl

ier

the

lip

id h

yd

rop

erod

ixe

lev

els

reac

hed

max

imu

m. O

xid

ativ

e d

amag

e is

rep

aire

d w

ith

in

15

day

s. V

itam

in E

pre

ven

ted

lip

id p

ero

xid

atio

n

bu

t no

t 8

-hyd

roxy

-2'-

deo

xyg

uan

osi

ne

form

atio

n.

Th

e h

igh

est

do

se -

red

uce

d b

od

y w

eig

ht

gai

n,

mil

d a

nem

ia,

and

leu

ko

cyto

sis.

Liv

er i

nju

ry -

mo

der

ate

to s

ever

e at

th

e h

igh

est

do

se,

to v

ery

slig

ht

at t

he

10

0 m

g/k

g d

ose

. M

agli

gn

ant

and

ben

ign t

um

ors

at

hig

hes

t d

ose

, d

ecre

asin

g

amo

un

ts o

f b

enig

n t

um

ors

.

An

imal

s w

ere

pre

trea

ted

wit

h s

afro

le a

nd

iso

safr

ole

bef

ore

in

ject

ed w

ith

2-

acet

amid

ofl

uo

ren

e in

crea

sed

th

e ac

tiv

ity

of

liv

er m

icro

som

al m

ater

ial

for

hy

dro

xy

lati

on

of

2-a

ceta

mid

ofl

uo

ren

e, w

hic

h c

an i

nh

ibit

liv

er

carc

ino

gen

esis

.

Die

t o

f 0

.25

%

iso

safr

ole

fo

r 1

4

day

s

Rat

s: s

ing

le 0

,

25

0,

50

0,1

000

mg

/kg v

ia i

p

inje

ctio

n,

wit

h

on

e g

roup

giv

en

30

0 m

g/k

g

Vit

amin

E

Rat

s, a

du

lt:

10

0,

50

0,

10

00

, 2

00

0

mg

/kg o

ver

tw

o

yea

rs,

or

5,

25

,

50

, an

d 1

00

mg

/kg

/day

.

Rat

s, i

n v

ivo

stu

dy

: 10

0

mg

/kg b

w,

In

vit

ro s

tud

y:

100

mg

/kg b

w,

and

30

0 m

g/k

g b

w;

Ham

ster

s: 2

00

mg

/kg b

w

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Mal

e W

ista

r

alb

ino

rat

s

Mal

e

Sp

rag

ue-

Daw

ley

rat

s

(20

0 g

)

Osb

orn

e-

Men

del

rat

s

Mal

e al

bin

o

Wis

tar

rats

(60

-120

g)

and

Sy

rian

go

lden

ham

ster

s

(10

0 g

)

Lak

e, B

. G

. &

D. V

. P

ark

e.

Ind

uct

ion

of

ary

l

hy

dro

carb

on

hyd

roxy

lase

in

var

iou

s ti

ssu

es o

f th

e ra

t b

y

met

hy

len

edio

xy

ph

eny

l

com

pou

nd

s. B

ioch

em.

J.

13

0:

86.

Liu

, T

. Y

., C

. C

. C

hen

, C

. L

.

Ch

en,

and

C.

W. C

hi.

Saf

role

-in

du

ced

Ox

idat

ive

Dam

age

in t

he

Liv

er o

f

Sp

rag

ue-

Daw

ley

Rat

s. F

oo

d

and

Ch

emic

al T

ox

ico

logy

37

: 69

7-7

02

.

Lo

ng

, E

.L.,

A.A

. N

elso

n,

O.G

. F

itzh

ugh

and

W.H

.

Han

sen

. L

iver

tu

mo

urs

pro

du

ced

in

rat

s b

y f

eed

ing

safr

ole

. A

rch

ives

of

Pat

ho

logy

75

: 5

95

-604

.

Lo

tlik

ar,

P. D

. &

Was

serm

an,

M.

B. E

ffec

ts

of

safr

ole

an

d i

sosa

fro

le

pre

trea

tmen

t o

n N

- an

d r

ing

-

hy

dro

xy

lati

on

of

2-

acet

amid

ofl

uo

ren

e b

y t

he

rat

and

ham

ster

. B

ioch

em.

J.

12

9:

937

-94

3.

19

72

19

99

19

63

19

72

87

Pre

gn

ancy

alt

ered

th

e b

ind

ing

of

safr

ole

to

DN

A b

y i

ncr

easi

ng

th

e

add

uct

bin

din

g t

o l

iver

an

d k

idn

ey

DN

A b

y 2

.3-3

.5 t

imes

.

2 m

ajo

r ad

du

cts

form

ed i

n t

he

N2

po

siti

on

of

gu

anin

e w

ith

saf

role

an

d

1'-

hyd

roxy

safr

ole

Saf

role

tes

ted

neg

ativ

e fo

r

un

sch

edu

led

DN

A s

yn

thes

is a

nd

is

no

t m

uta

gen

etic

in

bac

teri

a. 1

'-

hy

dro

xy

safr

ole

was

als

o

no

nm

uta

gen

etic

.

Saf

role

id

enti

fied

as

a ca

rcin

og

en,

no

n-m

uta

gen

ic,

wit

h l

ess

than

70

rev

erta

nt

colo

nie

s o

n a

pet

ri d

ish

per

10

00

mic

rog

ram

s o

f ch

emic

al

inco

rpo

rate

d i

n t

he

pla

te a

nd

<0

.01

rev

erta

nts

per

nm

ol

per

pla

te.

Use

d

stra

ins

TA

100

, T

A1

53

5, T

A1

53

7,

and

TA

98

.

On

e ti

me

do

se o

f

97

mg

/kg

to

pre

gn

ant

and

no

n-p

reg

nan

t

mic

e

Saf

role

Sta

nd

ard

Saf

role

stan

dar

d

ICR

mic

e

Mic

e

Un

sch

edu

le

d D

NA

syn

thes

is

assa

ys

in

HeL

a ce

lls

Sa

lmon

ella

/

mic

roso

me

test

Lu

, L

.J.,

et

al.

Dif

fere

nce

s in

th

e

cov

alen

t b

ind

ing

of

ben

zo(a

)py

ren

e,

safr

ole

, 1

’-h

yd

rox

ysa

fro

le a

nd

4-

amin

ob

iph

eny

l to

DN

A o

f p

reg

nan

t

and

no

n-p

reg

nan

t m

ice.

Can

cer

Let

ters

31

: 43

-52

.

Lu

, L

.J.,

et

al.

32

P-P

ost

-lab

elli

ng

assa

y i

n m

ice

of

tran

spla

cen

tal

DN

A

dam

age

ind

uce

d b

y t

he

env

iron

men

tal

carc

ino

gen

s sa

fro

le,

4-

amin

ob

iph

eny

l, a

nd

ben

zo(a

)py

ren

e.

Can

cer

Res

. 4

6:

304

6-3

05

4.

Mar

tin

, C

.N.,

A.C

. M

c D

erm

id a

nd

R.L

. G

arn

er.

Tes

tin

g o

f k

no

wn

carc

ino

gen

s an

d n

on

carc

ino

gen

s fo

r

thei

r ab

ilit

y t

o i

ndu

ce u

nsc

hed

ule

d

DN

A s

yn

thes

is i

n H

eLa

cell

s. C

ance

r

Res

earc

h 3

8:

26

21

-262

7.

McC

ann

, J.

et

al.

Det

ecti

on

of

carc

ino

gen

s as

mu

tag

ens

in t

he

Sal

mo

nel

la/m

icro

som

e te

st:

Ass

ay o

f

30

0 c

hem

ical

s. P

roce

edin

gs

of

the

Nat

ion

al A

cad

emy

of

Sci

ence

of

the

US

A 7

2 (

12

): 5

135

-513

9.

19

86

19

86

19

78

19

75

88

Saf

role

tes

ted

po

siti

ve

for

gen

e

mu

tati

on

ass

ay.

Fo

un

d l

iver

can

cer

in 6

1%

(v

. co

ntr

ol

of

24

%)

in m

ales

an

d 1

3%

in

fem

ales

.

Liv

er c

ance

r in

67

% (

v. 26

% c

on

tro

l),

lun

g a

den

om

as i

n 1

4%

fo

r bo

th

com

pou

nd

s. A

t b

oth

do

ses,

liv

er c

ance

r

in 6

8%

of

the

mic

e. L

ung

ad

eno

mas

wer

e fo

un

d i

n 5

% f

or

safr

ole

an

d 1

0%

in 1

'-hy

dro

xy

safr

ole

. L

iver

ca

nce

r in

55

% by

1'-

hyd

roxy

safr

ole

.

Saf

role

did

no

t in

du

ce u

nsc

hed

ule

d

DN

A s

yn

thes

is,

may

be

fro

m t

he

low

met

abo

liza

tio

n o

f 1

'-h

yd

roxy

safr

ole

.

Hy

dro

xy

chav

ico

l is

an

in

term

edia

te

met

abo

lite

of

safr

ole

an

d m

ore

to

xic

than

saf

role

as

it c

ause

s li

ver

cel

lula

r

mit

och

ond

ria

to d

ysf

un

ctio

n a

nd

cau

ses

gen

erat

ion

of

reac

tiv

e o

xyg

en s

pec

ies.

Mic

e: s

afro

le a

t

40

0 m

g/k

g t

en

tim

es &

23

00

or

46

00

mg

/kg

;

1'h

yd

rox

ysa

fro

le

at 8

1 m

g/k

g, 45

mg

/kg,

& 3

90

0

mg

/kg.

Rat

s:

10

00

, 1

10

0,

12

00

mg

/kg

A s

ing

le d

ose

of

20

0 o

r 1

00

0

mg

/kg

1 m

M o

f sa

fro

le

in 1

x 1

0^6

cell

s/m

L

Sta

nd

ard

Saf

role

stan

dar

d

Saf

role

stan

dar

d

In v

ivo

mam

mal

ia

n c

ell

gen

oto

xic

i

ty a

ssay

:

gen

e

mu

tati

on

CD

-1

mic

e (1

.4

g:

Day

1;

3.5

g:

Day

8;

7 g

:

Day

15

;

13

g:

Day

22

)

Mal

e

Fis

cher

-

34

4 r

ats

Mal

e

F3

44

/Jcl

(20

0-2

40

g)-

hep

ato

cyt

es w

ere

iso

late

d

Mih

r, B

., L

. B

ow

ers,

an

d W

.J.

Cas

par

y.

Ass

ays

for

the

ind

uct

ion

of

gen

e m

uta

tio

ns

at t

he

thy

mid

ine

kin

ase

locu

s in

L51

78

Y m

ou

se

lym

pho

ma

cell

s in

cu

ltu

re.

In:

Ash

by

and

de

Ser

res

(ed

s.).

Ev

alu

atio

n o

f

Sh

ort

-ter

m T

ests

fo

r C

arci

no

gen

s.

Els

evie

r, N

.Y.:

555

-56

8.

Mil

ler,

C.,

A.B

. S

wan

son

, D

.H.

Ph

illi

ps,

T.L

. F

letc

her

, A

. L

iem

, an

d

J.A

. M

ille

r. S

tru

ctu

re-a

ctiv

ity

stu

die

s

of

the

carc

ino

gen

icit

ies

in t

he

mo

use

and

rat

of

som

e n

atu

rall

y o

ccu

rrin

g

and

sy

nth

etic

alk

eny

lben

zen

e

der

ivat

ives

rel

ated

to

saf

role

an

d

estr

ago

le.

Can

cer

Res

. 4

3:

112

4-

11

34

.

Mir

sali

s, J

.C.,

K.

Ty

son,

and B

.E.

Bu

tter

wo

rth

. D

etec

tio

n o

f g

eno

tox

ic

carc

ino

gen

s in

th

e in

viv

o –

in

vit

ro

hep

ato

cyte

DN

A r

epai

r as

say

.

En

vir

on

. M

uta

gen

4:

55

3-5

62

.

Nak

agaw

a Y

., T

. S

uzu

ki,

K.

Nak

ajim

a, H

. Is

hii

, A

. O

gat

a.

Bio

tran

sfo

rmat

ion

an

d c

yto

tox

ic

effe

cts

of

hyd

roxy

chav

ico

l, a

n

inte

rmed

iate

of

safr

ole

met

abo

lism

, in

iso

late

d r

at h

epat

ocy

tes.

Ch

emic

o-

Bio

log

ical

In

tera

ctio

ns

18

0:

89

-97

.

19

85

19

83

19

82

20

09

89

Nin

hyd

rin

-po

siti

ve

bas

ic m

ater

ials

wer

e

excr

eted

in

th

e u

rin

e.

Saf

role

dec

reas

ed t

he

amo

un

t o

f g

lyco

gen

in t

he

liv

er.

Ad

eny

late

cy

clas

e an

d 3

:5-

cycl

ic A

MP

may

co

ntr

ibu

te, b

ut

no

rad

ren

alin

e d

id n

ot

incr

ease

bip

hen

yl

2-

or

4-h

yd

rox

yla

ses.

Incr

ease

d c

yto

chro

me

b5

an

d c

han

ged

th

e

abso

rpti

on

sp

ectr

um

, w

hic

h c

ou

ld r

esu

lt

fro

m b

iosy

nth

esis

of

a h

epat

ic

mic

roso

mal

hae

mo

pro

tein

. T

he

new

abso

rpti

on

sp

ectr

um

was

no

t o

bse

rved

wh

en t

hio

acet

amin

e w

as a

dd

ed t

o t

he

safr

ole

in

du

ctio

n.

2 m

ajo

r ad

du

cts

form

ed i

n t

he

N2 p

osi

tio

n

of

gu

anin

e w

ith

saf

role

an

d 1

'-

hy

dro

xy

safr

ole

Sin

gle

do

se

of

75

-300

mg

/kg b

w

12

5 m

g/k

g

per

day

Saf

role

stan

dar

d

Mal

e

Sp

rag

ue

Daw

ley

rats

(2

00

-

50

0 g

)

Rat

mo

del

Rat

mo

del

Mic

e

mo

del

Osw

ald

, E

. O

., L

. F

ish

bei

n &

B.J

.

Co

rbet

t. M

etab

oli

sm o

f n

atu

rall

y

occ

urr

ing

pro

pen

ylb

enze

ne

der

ivat

ives

.

I. C

hro

mat

og

rap

hic

sep

arat

ion

of

nin

hy

dri

n-p

osi

tiv

e m

ater

ials

of

rat

uri

ne,

Jou

rnal

of

Ch

rom

ato

gra

ph

y 4

5:

437

-445

.

Par

ke,

D.

V.

& H

. R

ahm

an. T

he

ind

uct

ion

of

hep

atic

mic

roso

mal

enzy

mes

by

saf

role

. B

ioch

em.

J. 1

19

:

53

P.

Par

ke,

D.

V.

& H

. R

ahm

an.

Ind

uct

ion

of

a n

ew h

epat

ic m

icro

som

al h

aem

op

rote

in

by

saf

role

an

d i

sosa

fro

le.

Bio

chem

. J.

12

3:

9P

.

Ph

illi

ps,

D.,

M.V

. R

eddy

and

K.

Ran

der

ath

. 3

2P

- P

ost

lab

elli

ng

an

aly

sis

of

DN

A a

dd

uct

s fo

rmed

in

th

e li

ver

s o

f

anim

als

trea

ted

wit

h s

afro

le,

estr

ago

le

and

oth

er n

atu

rall

yo

ccu

rin

g

alk

eny

lben

zen

es.

II.

New

bo

rn m

ale

B6

C3

F1

mic

e. C

arci

no

gen

esis

5:

162

3-

16

28

.

19

69

19

70

19

71

19

84

90

2 m

ino

r ad

du

cts

form

ed i

n t

he

N6

po

siti

on

of

aden

ine

wit

h s

afro

le

and

1'-h

yd

roxy

safr

ole

Saf

role

tes

ted

po

siti

ve

in b

oth

Saf

role

tes

ted

po

siti

ve

for

the

Am

es t

est,

cel

l tr

ansf

orm

atio

n,

deg

ran

ula

tio

n,

and

seb

aceo

us-

gla

nd

sup

pre

ssio

n a

nd

tes

ted

neg

ativ

e fo

r te

traz

oli

um

red

uct

ion

and

th

e im

pla

nt

test

s.

2 m

ajo

r ad

du

cts

form

ed i

n t

he

N2

po

siti

on

of

gu

anin

e w

ith

saf

role

and

1'-h

yd

roxy

safr

ole

. A

fter

a

sin

gle

do

se o

f 10

mg

of

safr

ole

,

on

e ad

du

ct f

orm

ed i

n 1

40

,000

nu

cleo

tid

es.

RA

L x

10

^7

val

ues

for

safr

ole

wer

e 4

91

+/-

12

9 f

or

10

mg

and

11

4 +

/- 5

0 f

or

the

2 m

g

do

se.

12

mic

rom

ole

s/

mo

use

of

1'-

[2',3

'-3H

]-

hy

dro

xy

safr

ole

via

i.p

.

inje

ctio

n.

2 m

g f

or

DN

A

bin

din

g s

tud

ies

or

10 m

g f

or

add

uct

per

sist

ence

stu

die

s. M

ice:

80

mg

/kg

or

40

0 m

g/k

g.

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Fem

ale

CD

-1

mic

e, 8

-10

wee

ks

old

(30

g)

E.

coli

an

d

Sa

cch

aro

myc

es c

erev

isia

e

in v

itro

assa

ys

Am

es t

est

wit

h

Sa

lmon

ella

typ

him

uri

um

;

Rab

in's

tes

t

and

oth

ers

Fem

ale

CD

-1

mic

e (2

5 g

)

Ph

illi

ps,

D.H

., J

.A. M

ille

r, E

.C.

Mil

ler,

and

B. A

dam

s. N

2 a

tom

of

gu

anin

e an

d

N6

of

aden

ine

resi

du

es a

t si

tes

for

cov

alen

t b

ind

ing

of

met

abo

lica

lly

acti

vat

ed 1

’- h

yd

roxy

safr

ole

to

mo

use

liv

er D

NA

in

viv

o.

Can

cer

Res

. 4

1:

26

24

-26

71

.

Po

irie

r, L

.A.

and

F.

J. d

e S

erre

s. I

nit

ial

Nat

ion

al C

ance

r In

stit

ute

stu

die

s o

n

mu

tag

enes

is a

s a

pre

-scr

een

fo

r

chem

ical

car

cin

og

ens:

an

ap

pra

isal

. J.

Nat

l. C

ance

r In

st.

62

: 9

19

-926

.

Pu

rch

ase,

I.

F.

H.

et a

l. A

n e

val

uat

ion

of

6 s

ho

rt-t

erm

tes

ts f

or

det

ecti

ng

org

anic

ch

emic

al c

arci

no

gen

s, B

r. J

.

Can

cer

37

: 8

73

-90

3.

Ran

der

ath

, K

., R

.E.

Hag

lund

, D

.H.

Ph

illi

ps

and

M.V

. R

eddy

. 3

2P

-

Po

stla

bel

lin

g a

nal

ysi

s o

f D

NA

ad

du

cts

form

ed i

n t

he

liv

ers

of

anim

als

trea

ted

wit

h s

afro

le,

estr

ago

le a

nd

oth

er

nat

ura

lly

-occ

urr

ing

alk

eny

lben

zen

es.

I.

Ad

ult

fem

ale

CD

-1 m

ice.

Car

cin

og

enes

is 5

: 1

613

-162

2.

19

81

19

79

19

78

19

84

91

Mic

e th

at o

nly

dra

nk c

ola

-dri

nk

s

dev

elo

ped

100

-200

DN

A a

dd

uct

s in

10

^9

DN

A n

ucl

eoti

des

, w

ith

saf

role

5-6

% o

f

the

add

uct

s an

d m

yri

stic

in 5

0-8

0%

. A

t 8

wee

ks,

saf

role

re

pre

sen

ted

5-6

% o

f th

e

tota

l ad

du

cts.

Fo

r m

yri

stic

in,

nu

tmeg

,

and

mac

e, s

afro

le a

dd

uct

s re

pre

sen

ted

3.5

-8.5

% o

f th

e to

tal

addu

cts.

Dih

yd

rosa

fro

le i

nje

stio

n r

esu

lted

in

neo

pla

sms

of

the

fore

sto

mac

h.

Saf

role

and

iso

safr

ole

did

no

t h

ave

sto

mac

h

tum

ors

, bu

t sa

fro

le d

id i

ncr

ease

th

e

inci

den

ces

of

liv

er c

arci

no

mas

.

A d

ose

of

20 m

g/k

g o

f sa

fro

le d

ou

ble

d t

he

mea

n s

leep

ing

tim

e co

mp

ared

to

th

e

con

tro

l w

hen

ad

ded

to

th

e sl

eep

ing

tab

let

com

pou

nd

sod

ium

pen

tob

arb

ital

. S

afro

le

did

no

t si

gn

ific

antl

y a

ffec

t th

e sl

eep

ing

tim

e in

ass

oci

atio

n w

ith

eth

ano

l.

In t

he

mic

e th

at r

ecei

ved

4.5

0 g

/kg

bw

, 1

0

ou

t o

f 3

0 d

ied

and

th

ree

mic

e h

ad l

un

g

tum

ors

. T

he

mic

e th

e re

ceiv

ed 0

.90

g/k

g

bw

, 3

ou

t o

f 3

0 d

ied

and

6 m

ice

had

lun

g

tum

ors

.

Mic

e :c

ola

dri

nk

s

inst

ead

of

wat

er (

for

4 o

r 8

wee

ks)

, m

ice

giv

en a

sin

gle

10

mg

do

se o

f m

yri

stic

in,

nu

tmeg

, o

r m

ace

via

gas

tric

in

tub

atio

n.

46

4 m

g/k

g o

f sa

fro

le

& d

ihy

dro

safr

ole

:

21

5 m

g/k

g o

f

iso

safr

ole

; 1

,11

2

pp

m s

afro

le,

1,4

00

pp

m d

ihy

dro

safr

ole

,

& 5

17

pp

m

iso

safr

ole

.

10

or

20

mg

/kg v

ia

i.p

. in

ject

ion

alo

ng

wit

h s

od

ium

pen

tob

arb

ital

an

d

10

0 m

g/k

g v

ia i

.p.

inje

ctio

n o

f et

han

ol

Ov

er 2

4 w

eek

s,

mic

e re

ceiv

ed a

to

tal

of

4.5

0 o

r 0.9

0 g

/kg

bw

of

safr

ole

ov

er

12

in

ject

ion

s.

Saf

role

Sta

nd

ard

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Fem

ale

ICR

mic

e, 6

-7

wee

ks

Hy

bri

d

mic

e

Fem

ale

wh

ite

mic

e

(22

-40 g

)

A/H

e

mic

e, 6

-8

wee

ks

old

(18

-

20

g)

Ran

der

ath

, K

.P.,

K.L

. P

utm

an,

and

E. R

and

erat

h.

Fla

vo

r

con

stit

uen

ts i

n c

ola

dri

nk

s

ind

uce

hep

atic

DN

A a

dd

uct

s in

adu

lt a

nd

fet

al m

ice.

Bio

chem

ical

Bio

ph

ysi

cal

Res

earc

h C

om

mun

icat

ion

s 1

92

(1):

61

-68

.

Reu

ber

, M

. D

. N

eop

lasm

s o

f th

e

fore

sto

mac

h i

n m

ice

ing

esti

ng

dih

yd

rosa

fro

le,

Dig

esti

on

, 1

9,

42

-47

Set

o,

T. A

. &

W. K

eup.

Eff

ects

of

alk

ylm

eth

oxy

ben

zen

e an

d

alk

ylm

eth

yle

ned

ioxy

ben

zen

e

esse

nti

al o

ils

on

pen

tob

arb

ital

and

eth

ano

l sl

eep

ing

tim

e. A

rch

Inte

rnd

e P

har

mac

od

yn

amie

et

de

ther

apie

18

0:

23

2-2

40.

Sto

ner

, G

. D

. et

al.

Tes

t fo

r

carc

ino

gen

icit

y o

f fo

od

add

itiv

es

and

ch

emo

ther

apeu

tic

agen

ts b

y

the

pu

lmon

ary

tu

mo

r re

spo

nse

in

stra

in A

mic

e, C

ance

r R

esea

rch

33

: 30

69

-30

85

.

19

93

19

79

19

69

19

73

92

Saf

role

was

neu

tral

or

wea

kly

po

siti

ve

in

the

Am

es t

est.

1'-

hy

dro

xy

safr

ole

was

mu

tag

enic

fo

r st

rain

TA

100

. T

ox

icit

y

incr

ease

d w

ith

ad

dit

ion

of

NA

DP

H-

fort

ifie

d r

at l

iver

mic

roso

mes

an

d

cyto

sol.

Pre

vio

us

stu

die

s w

ith

po

siti

ve

mu

tag

enic

pro

per

ties

use

d a

pre

incu

bat

ion

per

iod,

wh

ich

may

be

hav

e

crea

ted

met

abo

lite

s.

No

rat

s d

ied

ou

t o

f si

x t

este

d f

or

safr

ole

,

bu

t an

av

erag

e o

f 2

.5 m

acro

sco

pic

liv

er

lesi

on

s w

ere

ob

serv

ed.

Th

e ra

ts l

ost

wei

gh

t an

d w

ere

in p

oo

r co

nd

itio

n a

t th

e

end

of

the

exp

erim

ent.

Saf

role

in

hib

its

CY

P1

A2

, C

YP

2A

6,

CY

P2

D6,

CY

P2E

1, an

d C

YP

3A

4

Saf

role

an

d/o

r m

etab

oli

tes

cro

ssed

th

e

pla

cen

ta a

nd

was

del

iver

ed t

o i

nfa

nts

via

lact

atio

n,

bu

t y

ou

ng

mic

e h

ad h

ad m

uch

hig

her

su

rviv

al r

ate

(>9

0%

) w

hen

in

ute

ro a

nd

nu

rsin

g f

rom

mo

ther

s

rece

ivin

g s

afro

le c

om

par

ed t

o w

ean

ed

rats

th

at r

ecei

ved

dir

ect

safr

ole

do

ses.

Sa

lmo

nel

la

typ

him

uri

um

stra

ins

TA

100

,

TA

15

35

, an

d

TA

98

65

0 m

g/k

g b

w

via

gav

age,

or

1/3

of

the

LD

50

of

safr

ole

12

0 m

icro

gra

m

per

g o

f b

w t

o

pre

gn

ant

mic

e,

lact

atin

g

mo

ther

s, a

nd

4

wee

k o

ld

off

spri

ng

Saf

role

stan

dar

d

Saf

role

stan

dar

d

Saf

role

stan

dar

d

In v

itro

Sal

mo

nel

l

a re

ver

se

mu

tati

on

assa

y

(Am

es

test

)

Osb

orn

e-

Men

del

or

Sh

erm

an

rats

(1

80

-

35

0 g

)

Esc

her

ich

ia c

oli

-

exp

ress

ed

hu

man

P4

50

Fem

ale

C5

7B

L/6

J

and

mal

e

C3

HeB

/F

eJ m

ice,

8

wee

ks

Sw

anso

n,

A.B

., D

.D.

Ch

amb

liss

, J.

C.

Bla

nq

uis

t, E

.C.

Mil

ler

and

J.A

.

Mil

ler.

Th

e m

uta

gen

icit

ies

of

safr

ole

,

estr

ago

le,

tran

s-an

eth

ole

an

d s

om

e o

f

thei

r k

no

wn

or

po

ssib

le m

etab

oli

tes

for

Sal

mo

nel

la t

hy

ph

imu

riu

m

mu

tan

ts.

Mu

tat.

Res

. 6

0:

143

-15

3.

Tay

lor,

J.M

.,P

.M. Je

nn

er a

nd

W.I

.

Jon

es.

A c

om

par

iso

n o

f th

e to

xic

ity

of

som

e al

lyl,

pro

pen

yl

and

pro

py

l

com

pou

nd

s in

th

e ra

ts.

Tox

ico

logy

and

Ap

pli

ed P

har

mac

olo

gy

6:

37

8-

38

7.

Uen

g, Y

-F.,

et

al.

Inh

ibit

ion

of

hu

man

cy

toch

rom

e P

45

0 e

nzy

mes

by

the

nat

ura

l h

epat

oto

xin

saf

role

. F

ood

and

Ch

emic

al T

ox

ico

logy

43

: 70

2-

71

2.

Ves

seli

no

vit

ch,

S.D

., K

.V. R

ao, an

d

N.

Mih

ailo

vic

h. T

ran

spla

cen

tal

and

lact

atio

nal

car

cin

og

enes

is b

y s

afro

le.

Can

cer

Res

. 3

9:

437

8-4

38

0.

19

79

19

64

20

05

19

79

93

Ex

amin

ed t

he

ov

er 7

yea

rs.

Fo

un

d s

om

e ti

ssu

e in

jury

in

the

beg

inn

ing

of

the

exp

erim

ent,

bu

t co

ncl

ud

ed t

he

do

gs

may

hav

e ad

apte

d t

o t

he

con

tin

uo

us

inta

ke

of

safr

ole

.

2 m

ajo

r ad

du

cts

form

ed i

n t

he

N2 p

osi

tio

n o

f g

uan

ine

wit

h

safr

ole

an

d 1

'-hy

dro

xy

safr

ole

.

20

% h

ad h

epat

om

as.

Am

es

test

: 1

'-ac

eto

xy

safr

ole

, sa

fro

le-

2',3

'-o

xid

e, 1

'-ac

eto

xy

safr

ole

,

and

1'-o

xy

safr

ole

are

dir

ectl

y

mu

tag

enic

in

mu

tag

enic

in

Sal

mo

nel

la s

trai

n T

A15

35

.

Saf

role

ox

ide

inh

ibit

s

ang

iog

enes

is i

nd

uce

d b

y

can

cer

cell

s b

oth

in

vit

ro a

nd

in v

ivo

.

Bro

msu

lfal

ein

excr

etio

n c

urv

e,

solu

ble

en

zym

e, t

he

lip

id a

nd

gly

cog

en

of

the

liv

er,

and

tnit

ro-r

edu

ctas

e

0.1

mic

rom

ole

s o

f

1'-

hyd

roxy

safr

ole

per

g o

f b

od

y

wei

gh

t

10

or

2 m

g p

er 1

00

g o

f b

od

y w

eig

ht

via

i.p

. in

ject

ion

;

Sa

lmon

ella

str

ain

TA

10

0

Sy

nth

esi

zed

fro

m

stan

dar

ds

Sy

nth

esi

zed

fro

m

stan

dar

ds

Saf

role

stan

dar

d

syn

thes

iz

ed t

o

safr

ole

ox

ide

Do

g m

od

el

Mal

e h

yb

rid

mic

e

B6

C3

F1

Mal

e C

D

rats

; C

D-1

mic

e, a

nd

mal

e

ham

ster

s;

Am

es t

est

Vas

cula

r

end

oth

elia

l

cell

s, A

54

9

lun

g c

ance

r

cell

s, r

at

aort

a fr

om

mal

e

Sp

rag

ue-

Daw

ley

rat

s

Wei

nb

erg

, M

. S

. &

S. S

. S

tern

ber

g.

Eff

ect

of

chro

nic

saf

role

ad

min

istr

atio

n

on

hep

atic

en

zym

es a

nd

fu

nct

ion

al

acti

vit

y i

n d

og

s. T

ox

ico

logy

and

Ap

pli

ed

Ph

arm

aco

log

y 8

: 2

.

Wis

eman

, R

.W.,

et

al.

Fu

rth

er

char

acte

riza

tio

n o

f th

e D

NA

ad

du

cts

form

ed b

y e

lect

rop

hil

ic e

ster

s o

f th

e

hep

ato

carc

ino

gen

s 1

'-h

yd

roxy

safr

ole

an

d

1'-

hyd

roxy

estr

ago

le i

n v

itro

an

d i

n t

he

mo

use

liv

er i

n v

ivo

, in

clu

din

g n

ew

add

uct

s at

C-8

an

d N

-7 o

f gu

anin

e

resi

du

es.

Can

cer

Res

. 4

5:

30

96

-310

5.

Wis

lock

i, P

.G., E

.C.

Mil

ler,

J.A

. M

ille

r,

E.C

. M

cCo

y,

and

H.S

. R

ose

nk

ran

z.

Car

cin

og

enic

an

d m

uta

gen

ic a

ctiv

itie

s o

f

safr

ole

, 1

'-h

yd

roxy

safr

ole

an

d s

om

e

kn

ow

n o

r po

ssib

le m

etab

oli

tes.

Can

cer

Res

. 3

7:

18

83

-18

91

.

Zh

ao,

J, J

. M

iao

, B

. Z

hao

, S

. Z

han

g, D

.

Yin

. S

afro

le o

xid

e in

hib

its

ang

iog

enes

is

by

ind

uci

ng

apo

pto

sis.

Vas

cula

r

Ph

arm

aco

log

y 4

3:

69

-74

.

19

66

19

85

19

77

20

05

94

Table C.3: Review of studies using chemical analysis of foods containing safrole.

R

esu

lts

Ex

amin

ed a

dif

fere

nt

met

abo

lic

pat

hw

ay

safr

ole

co

uld

un

der

go,

nam

ely

th

roug

h

ox

idat

ion

to

qu

ino

id m

etab

oli

tes

in l

iver

mic

roso

mes

in

a n

on

enzy

mat

ic p

roce

ss.

Th

e ‘t

ea’

mad

e in

th

is s

tud

y w

as i

nfu

sed

or

the

tea

bag

ste

eped

in

ho

t w

ater

fo

r

thir

ty m

inu

tes

as o

ppo

sed

to

th

e

trad

itio

nal

met

ho

d o

f d

eco

ctin

g, o

r

bo

ilin

g, th

e w

ho

le o

r ro

ugh

ly c

ho

pp

ed

roo

t. T

hey

dem

on

stra

ted

th

at h

erb

al

pre

par

atio

ns

of

sass

afra

s ra

ng

ed f

rom

har

mle

ss f

or

tea

mad

e fr

om

th

e le

aves

, to

con

tain

ing

92

.4%

saf

role

in

sas

safr

as o

ils.

Th

e ‘t

ea’

they

pre

par

ed h

ad s

afro

le

con

ten

t ra

ng

ing f

rom

0.0

9 t

o 4

.12

mg

per

cup

, o

r on

ly 6

.9 t

o 1

7.2

% o

f to

tal

safr

ole

pre

sen

t in

ro

ot.

Th

e sa

fro

le m

etab

oli

tes

dih

yd

roxy

chav

ico

l an

d e

ug

eno

l w

ere

fou

nd

in

th

e u

rin

e o

f n

on

-bet

el q

uid

chew

ers,

at

the

lev

els

of

no

t d

etec

ted

to

5.4

mic

rog

ram

s/m

g c

reat

inin

e, p

rob

ably

du

e to

sp

ices

in

th

e d

iet.

Co

nce

ntr

ati

on

s

0,

30

, 7

5,

15

0,

30

0

mg

/kg o

ne

tim

e.

Tes

ted

th

e u

rin

e

of

the

rats

an

d o

f

hu

man

su

bje

cts

wh

o c

hew

ed b

etel

qu

id b

etw

een

2 -

10

0 t

imes

a d

ay.

Ch

emic

al

An

aly

sis

HP

LC

,

UV

spec

tro

ph

oto

met

er

Liq

uid

Ch

rom

ato

gra

ph

y

HP

LC

So

urc

e

Saf

role

stan

dar

d

Po

wd

ere

d l

eav

es,

gu

mb

o

file

, ro

ot

bar

k,

roo

t

po

wd

er,

her

bal

cap

sule

mat

eria

l,

un

bre

we

d t

ea.

Saf

role

stan

dar

d

Art

icle

Bo

lto

n,

J.L

., e

t a.

Ev

iden

ce

that

4-a

lly

l-O

-qu

ino

nes

spo

nta

neo

usl

y r

earr

ang

e to

thei

r m

ore

ele

ctro

ph

ilic

qu

ino

ne

met

hid

es:

po

ten

tial

bio

acti

vat

ion

mec

han

ism

for

the

hep

ato

carc

ino

gen

safr

ole

. C

hem

Res

. T

ox

ico

l.

7:

443

-45

0.

Car

lso

n,

M.

and

R. D

.

Th

om

pso

n. L

iqu

id

Ch

rom

atog

rap

hic

Det

erm

inat

ion

of

Saf

role

in

Sas

safr

as-D

eriv

ed H

erb

al

Pro

du

cts.

Jo

urn

al o

f A

OA

C

Inte

rnat

ion

al 8

0 (

5):

102

3-

8.

Ch

ang,

M.J

.W., C

.Y. K

o,

R.F

. L

in &

L.L

. H

sieh

.

Bio

log

ical

mo

nit

ori

ng

of

env

iro

nm

ent

exp

osu

re t

o

safr

ole

an

d t

he

Tai

wan

ese

bet

el q

uid

ch

ewin

g.

Arc

h.

En

vir

on

. C

on

tam

. T

ox

ico

l.

43

: 43

2–

43

7.

Yea

r

19

94

19

97

20

02

95

S

afro

le c

on

ten

t o

f th

e h

erb

al d

rug

extr

acte

d b

y M

eOH

ran

ged

bet

wee

n

1.5

7 t

o 2

.76

mg

/g.

Aft

er a

on

e ho

ur

dec

oct

ion

, sa

fro

le c

on

ten

t w

as

bet

wee

n 0

.20

mg

/g a

nd

no

t

det

ecta

ble

. T

he

med

icin

e fo

rmu

las

con

tain

ed 0

.06

mg

/g o

f sa

fro

le t

o n

ot

det

ecta

ble

.

Dev

elo

ped

sim

ple

met

hod

to

an

aly

ze

soft

dri

nk

s; f

ou

nd

20

ou

t o

f 25

so

ft

dri

nk

s h

ad l

evel

s o

f sa

fro

le a

nd

/or

cis-

iso

safr

ole

ex

ceed

ed t

he

lim

it o

f 1

mic

rog

ram

/mL

set

by

reg

ula

tio

ns,

tho

ug

h t

he

safr

ole

all

cam

e fr

om

'nat

ura

l se

aso

nin

g e

xtr

acts

'.

Fo

un

d s

afro

le p

rese

nt

in a

lco

ho

lic

dri

nk

s w

ith

lev

els

fro

m 6

.6 m

g/l

to

abse

nt.

Det

erm

ined

HP

LC

had

a

hig

h r

eco

ver

y r

ate

of

esse

nti

al o

ils-

for

safr

ole

rec

ov

ery

ran

ged

fro

m

95

.6 t

o 1

00

%.

3 g

of

po

wd

ered

her

bal

dru

g

dec

oct

ed i

n w

ater

or

extr

acte

d w

ith

MeO

H.

A

trad

itio

nal

Ch

ines

e

med

icin

e fo

rmu

la

that

co

nta

ined

3 g

of

Asa

ri r

adix

was

dec

oct

ed.

So

ft d

rin

ks

con

tain

ed 3

-5 m

g/L

(pp

m)

of

safr

ole

.

HP

LC

Gas

Ch

rom

atog

rap

hy

HP

LC

wit

h

flu

ori

met

ri

c d

etec

tio

n

and

GC

-

MS

Asa

ri r

adix

et

rhiz

om

a

Co

mm

erci

al

soft

dri

nk

s,

app

le c

ider

,

and

roo

t b

eer

Co

mm

erci

al

alco

ho

lic

bev

erag

es

and

ess

enti

al

oil

s o

f

nu

tmeg

,

sass

afra

s,

cin

nam

on

,

and

an

ise

Ch

en, C

., D

. S

pri

ano

, T

.

Leh

man

n,

and B

. M

eier

.

Red

uct

ion

of

Saf

role

an

d

Met

hy

leu

gen

ol

in A

sari

rad

ix

et r

hiz

om

a b

y D

eco

ctio

n.

Fo

rsch

Kom

ple

men

tmed

16

:

16

2-1

66

.

Ch

oo

ng

, Y

.-M

. &

H.-

J. L

in.

A R

apid

and

Sim

ple

Gas

Ch

rom

atog

rap

hic

Met

ho

d f

or

Dir

ect

Det

erm

inat

ion

of

Saf

role

in

So

ft D

rin

ks.

”

Jou

rnal

of

Fo

od

and

Dru

g

An

aly

sis

9(1

): 2

7-3

2.

Cu

rró

, P

., G

. M

ical

i, a

nd

F.

Lan

uzz

a. D

eter

min

atio

n o

f β

-

asar

on

e, s

afro

le,

iso

safr

ole

,

and

an

eth

ole

in

alc

hoh

oli

c

dri

nk

s by

hig

h-p

erfo

rman

ce

liq

uid

ch

rom

atog

rap

hy.

Jou

rnal

of

Ch

rom

oto

gra

phy

40

4:

273

-27

8.

20

09

20

01

19

87

96

Was

hin

g a

nd d

ryin

g r

edu

ced

th

e sa

fro

le c

on

ten

t

by

any

wh

ere

fro

m 9

4 t

o 7

4%

. G

amm

a

irra

dia

tio

n r

edu

ced

th

e sa

fro

le c

on

ten

t b

y 9

7 t

o

86

%, o

r o

nly

to

saf

e le

vel

s o

f sa

fro

le a

t th

e h

igh

lev

els.

Mic

row

ave

rad

iati

on

red

uce

d t

he

safr

ole

con

ten

t 9

2 t

o 5

7%

, bu

t on

ly t

he

lon

ges

t ti

me

red

uce

d t

o s

afe

lev

els

and

th

e sa

mp

les

bu

rned

.

Bo

ilin

g t

he

seed

s fo

un

d b

oil

ing w

ho

le s

eed

fo

r

5 m

inu

tes

and

bo

ilin

g p

ow

der

ed s

eed

fo

r 1

min

ute

was

eff

ecti

ve

at r

edu

cin

g s

afro

le t

o s

afe

lev

els.

New

met

ho

d o

f d

eter

min

ing

saf

role

, eu

gen

ol,

and

4-a

lly

l-1

,2-d

imet

ho

xy

ben

zen

e. T

este

d

un

bre

wed

tea

an

d f

oun

d s

afro

le l

evel

s b

etw

een

29

9 a

nd 1

7,4

00 m

g/k

g.

Tes

ted

th

e re

cov

ery

rat

es o

f co

mp

oun

ds

usi

ng

GC

an

d r

eco

mm

end

s ad

op

tin

g t

he

met

hod

fo

r

qu

anti

tati

ve

det

erm

inat

ion

of

safr

ole

, w

hic

h h

as

bee

n a

dop

ted

by

th

e F

DA

.

12

0 g

of

bla

ck

pep

per

see

ds

wer

e w

ash

ed &

dri

ed,

bo

iled

,

exp

ose

d t

o

gam

ma

irra

dia

tio

n,

and

mic

row

ave

irra

dia

tio

n b

oth

in w

ho

le s

eed

and

po

wd

ered

seed

fo

rm.

Gas

Ch

rom

atog

rap

hy

SF

E w

ith

GC

-MS

Gas

Ch

rom

atog

rap

hy

Sp

ices

:

Bla

ck

pep

per

,

gin

ger

roo

t,

cum

in,

star

an

ise.

Un

bre

wed

sass

afra

s

tea,

com

pou

nd

stan

dar

ds

Saf

role

stan

dar

d

Far

ag,

S.E

.A. &

M. A

bo

-

Zei

d.

Deg

red

atio

n o

f th

e

nat

ura

l m

uta

gen

ic

com

pou

nd

saf

role

in

sp

ices

by

coo

kin

g a

nd

irr

adia

tio

n.

Nah

rung

41

(6

): 3

59

-361

.

Hei

kes

, D

.L. S

FE

wit

h G

C

and

MS

det

erm

inat

ion

of

safr

ole

an

d r

elat

ed

ally

lben

zen

es i

n s

assa

fras

teas

. Jo

urn

al o

f

Ch

rom

atog

rap

hic

Sci

ence

32

(7

): 2

53

-258

.

Lar

ry,

D.

Gas

Ch

rom

atog

rap

hic

Det

erm

inat

ion

of

Saf

role

and

Rel

ated

Co

mp

ou

nd

s in

No

nal

coho

lic

Bev

erag

es:

Co

llab

ora

tiv

e S

tudy

.

Jou

rnal

of

the

AO

AC

54

(4):

90

0-9

02

.

19

97

19

94

19

70

97

E

xtr

acts

by

MeO

H s

how

ed

safr

ole

pre

sen

t in

th

e b

ark

by

0.4

35

% (

w/w

). S

afro

le

mea

sure

d 0

.000

1%

(w

/w)

in

the

tea

mad

e b

y b

oil

ing

in

wat

er,

wh

ich

is

abo

ut

30

0

tim

es m

ore

saf

role

pre

sen

t in

the

met

han

ol

extr

acte

d

sam

ple

s.

Ex

trac

ted

sas

safr

as o

il u

sin

g

pet

rol,

wh

ich

was

evap

ora

ted

an

d e

xtr

acte

d

agai

n w

ith

MeO

H. O

ily

frac

tio

n w

as a

nal

yze

d t

o f

ind

19

com

po

nen

ts o

f sa

ssaf

ras

oil

.

2 m

ajo

r ad

du

cts

form

ed i

n

the

N2 p

osi

tio

n o

f gu

anin

e

wit

h s

afro

le a

nd

1'-

hy

dro

xy

safr

ole

.

Ex

trac

ted

2.0

1 g

of

bar

k

wit

h M

eOH

. E

xtr

acti

ng

5.0

1 g

of

bar

k w

ith

wat

er

by

bo

ilin

g,

incl

ud

ing

on

e

sam

ple

set

th

at w

as a

lso

extr

acte

d w

ith

MeO

H.

An

oth

er s

amp

le s

et w

as

spik

ed w

ith

50

mic

rog

ram

s o

f sa

fro

le

stan

dar

d a

nd

bo

iled

in

wat

er

0.1

mic

rom

ole

s o

f 1

'-

hy

dro

xy

safr

ole

per

g o

f

bo

dy

wei

gh

t

HP

LC

GC

-MS

HP

LC

Cin

na

mo

mu

m

caro

lin

ens

e tr

un

k

bar

k

Po

wd

ered

roo

t b

ark

Sy

nth

esiz

ed f

rom

stan

dar

ds

Rey

ner

tso

n,

K.

A., M

. J.

Bal

ick

,

R.

Lee

, W

. R

ayno

r, Y

. P

elep

, an

d

E.

J. K

enn

elly

. A

Tra

dit

ion

al

Met

ho

d o

f C

inn

am

om

um

caro

lin

ense

Pre

par

atio

n

Eli

min

ates

Saf

role

fro

m a

Th

erap

euti

c P

ohn

pea

n T

ea.

Jou

rnal

of

Eth

nop

har

mac

olo

gy

10

2 (

2):

26

9-2

74.

Set

hi,

M.

L.,

G.

Su

bb

a R

ao, B

. K

.

Ch

ow

dh

ury

, J.

F.

Mo

rton

, &

G. J.

Kap

adia

. Id

enti

fica

tio

n o

f vo

lati

le

con

stit

uen

ts o

f S

ass

afr

as

alb

idu

m

roo

t o

il. P

hy

toch

emis

try

15

: 1

77

3-

17

75

.

Wis

eman

, R

.W.,

T.R

F

enn

ell,

J.A

Mil

ler,

an

d E

.C.

Mil

ler.

Fu

rth

er

char

acte

riza

tio

n o

f th

e D

NA

add

uct

s fo

rmed

by

ele

ctro

ph

ilic

este

rs o

f th

e h

epat

oca

rcin

og

ens

1'-

hy

dro

xy

safr

ole

an

d 1

'-

hy

dro

xy

estr

ago

le i

n v

itro

an

d i

n

the

mo

use

liv

er i

n v

ivo

, in

clu

din

g

new

ad

du

cts

at C

-8 a

nd

N-7

of

gu

anin

e re

sid

ues

. C

ance

r R

es.

45

:

30

96

-31

05

.

20

05

19

76

19

85

98

Appendix D

Abbreviations

Abbreviation Meaning

Bw Body Weight

CCP Center for Cherokee Plants

CDC Center for Disease Control and Prevention

CFR Code of Federal Regulations

EBCI Eastern Band of Cherokee Indians

FDA Food and Drug Administration

GIS Geographic Information System

HPLC High Performance-Liquid Chromatography

MCI Museum of the Cherokee Indian

mg/kg bw milligram/kilogram of body weight

ppm Parts Per Million

SCF Scientific Committee on Food (European Commission)

WHO World Health Organization

99

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Ames, B.N. & L.S. Gold. 2000. Paracelsus to parascience: the environmental cancer distraction. Mutation

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